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Immunohistochemistry Antibody Validation Report for Anti-Phospho-GSK3β (S9) Antibody (STJ90284)

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St John's Laboratory Ltd
St John's Laboratory Ltd

Constitutively active protein kinase that acts as a negative regulator in the hormonal control of glucose homeostasis, Wnt signaling and regulation of transcription factors and microtubules, by phosphorylating and inactivating glycogen synthase (GYS1 or GYS2), EIF2B, CTNNB1/beta-catenin, APC, AXIN1, DPYSL2/CRMP2, JUN, NFATC1/NFATC, MAPT/TAU and MACF1. Requires primed phosphorylation of the majority of its substrates. In skeletal muscle, contributes to insulin regulation of glycogen synthesis by phosphorylating and inhibiting GYS1 activity and hence glycogen synthesis. May also mediate the development of insulin resistance by regulating activation of transcription factors. Regulates protein synthesis by controlling the activity of initiation factor 2B (EIF2BE/EIF2B5) in the same manner as glycogen synthase. In Wnt signaling, GSK3B forms a multimeric complex with APC, AXIN1 and CTNNB1/beta-catenin and phosphorylates the N-terminus of CTNNB1 leading to its degradation mediated by ubiquitin/proteasomes. Anti-Phospho-GSK3β (S9)-http://www.stjohnslabs.com/phospho-gsk3b-s9-antibody Join our Antibody Validation Project - http://www.stjohnslabs.com/services/antibody-validation

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Figure: Immunohistochemical analysis of paraffin embedded Human uterus tissue. 1: GSK3β (phospho Ser9) Polyclonal Antibody was diluted at 1:200 (4 degree Celsius,overnight). 2: Sodium citrate pH 6.0 was used for antibody retrieval (>98 degree Celsius,20min). 3: Secondary antibody was diluted at 1:200 (room temperature, 30min). Negative control was used by secondary antibody only. Report Number 90284-a Host Rabbit Application IHC-P Clonality Polyclonal Model Number STJ90284 Clone ID NA Antibody Name Anti-Phospho-GSK3β (S9) antibody Testing Species HUMAN Testing Tissue UTERUS ANTIBODY VALIDATION REPORT a. (A small amount of distilled water was added into the incubation box to prevent evaporation of antibody). 249. Secondary antibody incubation a. Slides were washed 3 times, with PBS on a shaker for 5min. Shortly after the slides were dried the corresponding secondary antibody solution was added (HRP labelled), covering the tissues, and incubated at room temperature for 30min. b. 250. DAB staining a. Slides were washed 3 times, with PBS on a shaker for 5min. b. Shortly after, the slides were dried and fresh DAB staining buffer was added inside the circles. The staining time was adjusted under a microscope. Yellow-brown colour represented a positive result. Slides were washed with water to stop the staining. c. 251. Haematoxylin staining a. Haematoxylin was used to counter-staining for 1min, and then the slides were washed with water. 1% Hydrochloric acid and alcohol was added for several seconds and then washed with water. Ammonia was used to reveal blue colour, and then flushed with water. b. 252. Desolation and Clearing i. Slides were incubated sequentially into: 75% alcohol 5min, 85% alcohol 5min, Anhydrous ethanol - 5min, Anhydrous ethanol - 5min & Xylene - 5min. Shortly after slides were dried and neutral gum was used to seal the slides. ii. 253. Visualization a. Results were validated with microscope, and the slides were scanned. Paraffin-Embedded Immunohistochemistry Protocol 243. 244. Tissue processing a. Slides were incubated sequentially into Xylene; 15min – Xylene, 15min - Anhydrous ethanol, 5min - Anhydrous ethanol, 5min - 85% alcohol, 5min - 75% alcohol & 5min – wash in distilled water. b. 245. Antigen retrieval a. Tissue slides were incubated with citric acid (PH6.0) antigen retrieval buffer and microwaved for antigen retrieval (heated until boiled and then stopped heating) for 8min. Slides were then heated with medium power for 7min. During this process slides were kept from drying out. After cooling down at room temperature, slides were washed with PBS on shaker for 5min, repeated for 3 times. b. 246. Inhibition of endogenous peroxidase a. Slides were placed in 3% Hydrogen peroxide solution, and incubated for 10 min at room temperature without light exposure. Slides were then washed 3 times with PBS on a shaker for 5mins. b. 247. BSA Blocking a. Shortly after slides were dried, a PAP pen was used to draw circles around the tissue sections (and to prevent draining of the antibody solution). Inside the circles, BSA was used to cover the tissue evenly, blocking for 30min. b. 248. Primary antibody incubation After blocking solution was removed a 1:200 solution of primary antibody/PBS was added on the slide, and incubated overnight at 4°C. St John's Laboratory Ltd. www.stjohnslabs.com
Figure: Immunohistochemical analysis of paraffin embedded Human uterus cancer tissue. 1: GSK3β (phospho Ser9) Polyclonal Antibody was diluted at 1:200 (4 degree Celsius,overnight). 2: Sodium citrate pH 6.0 was used for antibody retrieval (>98 degree Celsius,20min). 3: Secondary antibody was diluted at 1:200 (room temperature, 30min). Negative control was used by secondary antibody only. Report Number 90284-b Host Rabbit Application IHC-P Clonality Polyclonal Model Number STJ90284 Clone ID NA Antibody Name Anti-Phospho-GSK3β (S9) antibody Testing Species HUMAN Testing Tissue UTERUS CANCER ANTIBODY VALIDATION REPORT a. (A small amount of distilled water was added into the incubation box to prevent evaporation of antibody). 238. Secondary antibody incubation a. Slides were washed 3 times, with PBS on a shaker for 5min. Shortly after the slides were dried the corresponding secondary antibody solution was added (HRP labelled), covering the tissues, and incubated at room temperature for 30min. b. 239. DAB staining a. Slides were washed 3 times, with PBS on a shaker for 5min. b. Shortly after, the slides were dried and fresh DAB staining buffer was added inside the circles. The staining time was adjusted under a microscope. Yellow-brown colour represented a positive result. Slides were washed with water to stop the staining. c. 240. Haematoxylin staining a. Haematoxylin was used to counter-staining for 1min, and then the slides were washed with water. 1% Hydrochloric acid and alcohol was added for several seconds and then washed with water. Ammonia was used to reveal blue colour, and then flushed with water. b. 241. Desolation and Clearing i. Slides were incubated sequentially into: 75% alcohol 5min, 85% alcohol 5min, Anhydrous ethanol - 5min, Anhydrous ethanol - 5min & Xylene - 5min. Shortly after slides were dried and neutral gum was used to seal the slides. ii. 242. Visualization a. Results were validated with microscope, and the slides were scanned. Paraffin-Embedded Immunohistochemistry Protocol 232. 233. Tissue processing a. Slides were incubated sequentially into Xylene; 15min – Xylene, 15min - Anhydrous ethanol, 5min - Anhydrous ethanol, 5min - 85% alcohol, 5min - 75% alcohol & 5min – wash in distilled water. b. 234. Antigen retrieval a. Tissue slides were incubated with citric acid (PH6.0) antigen retrieval buffer and microwaved for antigen retrieval (heated until boiled and then stopped heating) for 8min. Slides were then heated with medium power for 7min. During this process slides were kept from drying out. After cooling down at room temperature, slides were washed with PBS on shaker for 5min, repeated for 3 times. b. 235. Inhibition of endogenous peroxidase a. Slides were placed in 3% Hydrogen peroxide solution, and incubated for 10 min at room temperature without light exposure. Slides were then washed 3 times with PBS on a shaker for 5mins. b. 236. BSA Blocking a. Shortly after slides were dried, a PAP pen was used to draw circles around the tissue sections (and to prevent draining of the antibody solution). Inside the circles, BSA was used to cover the tissue evenly, blocking for 30min. b. 237. Primary antibody incubation After blocking solution was removed a 1:200 solution of primary antibody/PBS was added on the slide, and incubated overnight at 4°C. St John's Laboratory Ltd. www.stjohnslabs.com
Figure: Immunohistochemical analysis of paraffin embedded Human colon tissue. 1: GSK3β (phospho Ser9) Polyclonal Antibody was diluted at 1:200 (4 degree Celsius,overnight). 2: Sodium citrate pH 6.0 was used for antibody retrieval (>98 degree Celsius,20min). 3: Secondary antibody was diluted at 1:200 (room temperature, 30min). Negative control was used by secondary antibody only. Report Number 90284-c Host Rabbit Application IHC-P Clonality Polyclonal Model Number STJ90284 Clone ID NA Antibody Name Anti-Phospho-GSK3β (S9) antibody Testing Species HUMAN Testing Tissue COLON ANTIBODY VALIDATION REPORT a. (A small amount of distilled water was added into the incubation box to prevent evaporation of antibody). 227. Secondary antibody incubation a. Slides were washed 3 times, with PBS on a shaker for 5min. Shortly after the slides were dried the corresponding secondary antibody solution was added (HRP labelled), covering the tissues, and incubated at room temperature for 30min. b. 228. DAB staining a. Slides were washed 3 times, with PBS on a shaker for 5min. b. Shortly after, the slides were dried and fresh DAB staining buffer was added inside the circles. The staining time was adjusted under a microscope. Yellow-brown colour represented a positive result. Slides were washed with water to stop the staining. c. 229. Haematoxylin staining a. Haematoxylin was used to counter-staining for 1min, and then the slides were washed with water. 1% Hydrochloric acid and alcohol was added for several seconds and then washed with water. Ammonia was used to reveal blue colour, and then flushed with water. b. 230. Desolation and Clearing i. Slides were incubated sequentially into: 75% alcohol 5min, 85% alcohol 5min, Anhydrous ethanol - 5min, Anhydrous ethanol - 5min & Xylene - 5min. Shortly after slides were dried and neutral gum was used to seal the slides. ii. 231. Visualization a. Results were validated with microscope, and the slides were scanned. Paraffin-Embedded Immunohistochemistry Protocol 221. 222. Tissue processing a. Slides were incubated sequentially into Xylene; 15min – Xylene, 15min - Anhydrous ethanol, 5min - Anhydrous ethanol, 5min - 85% alcohol, 5min - 75% alcohol & 5min – wash in distilled water. b. 223. Antigen retrieval a. Tissue slides were incubated with citric acid (PH6.0) antigen retrieval buffer and microwaved for antigen retrieval (heated until boiled and then stopped heating) for 8min. Slides were then heated with medium power for 7min. During this process slides were kept from drying out. After cooling down at room temperature, slides were washed with PBS on shaker for 5min, repeated for 3 times. b. 224. Inhibition of endogenous peroxidase a. Slides were placed in 3% Hydrogen peroxide solution, and incubated for 10 min at room temperature without light exposure. Slides were then washed 3 times with PBS on a shaker for 5mins. b. 225. BSA Blocking a. Shortly after slides were dried, a PAP pen was used to draw circles around the tissue sections (and to prevent draining of the antibody solution). Inside the circles, BSA was used to cover the tissue evenly, blocking for 30min. b. 226. Primary antibody incubation After blocking solution was removed a 1:200 solution of primary antibody/PBS was added on the slide, and incubated overnight at 4°C. St John's Laboratory Ltd. www.stjohnslabs.com
Figure: Immunohistochemical analysis of paraffin embedded Human liver tissue. 1: GSK3β (phospho Ser9) Polyclonal Antibody was diluted at 1:200 (4 degree Celsius,overnight). 2: Sodium citrate pH 6.0 was used for antibody retrieval (>98 degree Celsius,20min). 3: Secondary antibody was diluted at 1:200 (room temperature, 30min). Negative control was used by secondary antibody only. Report Number 90284-d Host Rabbit Application IHC-P Clonality Polyclonal Model Number STJ90284 Clone ID NA Antibody Name Anti-Phospho-GSK3β (S9) antibody Testing Species HUMAN Testing Tissue LIVER ANTIBODY VALIDATION REPORT a. (A small amount of distilled water was added into the incubation box to prevent evaporation of antibody). 216. Secondary antibody incubation a. Slides were washed 3 times, with PBS on a shaker for 5min. Shortly after the slides were dried the corresponding secondary antibody solution was added (HRP labelled), covering the tissues, and incubated at room temperature for 30min. b. 217. DAB staining a. Slides were washed 3 times, with PBS on a shaker for 5min. b. Shortly after, the slides were dried and fresh DAB staining buffer was added inside the circles. The staining time was adjusted under a microscope. Yellow-brown colour represented a positive result. Slides were washed with water to stop the staining. c. 218. Haematoxylin staining a. Haematoxylin was used to counter-staining for 1min, and then the slides were washed with water. 1% Hydrochloric acid and alcohol was added for several seconds and then washed with water. Ammonia was used to reveal blue colour, and then flushed with water. b. 219. Desolation and Clearing i. Slides were incubated sequentially into: 75% alcohol 5min, 85% alcohol 5min, Anhydrous ethanol - 5min, Anhydrous ethanol - 5min & Xylene - 5min. Shortly after slides were dried and neutral gum was used to seal the slides. ii. 220. Visualization a. Results were validated with microscope, and the slides were scanned. Paraffin-Embedded Immunohistochemistry Protocol 210. 211. Tissue processing a. Slides were incubated sequentially into Xylene; 15min – Xylene, 15min - Anhydrous ethanol, 5min - Anhydrous ethanol, 5min - 85% alcohol, 5min - 75% alcohol & 5min – wash in distilled water. b. 212. Antigen retrieval a. Tissue slides were incubated with citric acid (PH6.0) antigen retrieval buffer and microwaved for antigen retrieval (heated until boiled and then stopped heating) for 8min. Slides were then heated with medium power for 7min. During this process slides were kept from drying out. After cooling down at room temperature, slides were washed with PBS on shaker for 5min, repeated for 3 times. b. 213. Inhibition of endogenous peroxidase a. Slides were placed in 3% Hydrogen peroxide solution, and incubated for 10 min at room temperature without light exposure. Slides were then washed 3 times with PBS on a shaker for 5mins. b. 214. BSA Blocking a. Shortly after slides were dried, a PAP pen was used to draw circles around the tissue sections (and to prevent draining of the antibody solution). Inside the circles, BSA was used to cover the tissue evenly, blocking for 30min. b. 215. Primary antibody incubation After blocking solution was removed a 1:200 solution of primary antibody/PBS was added on the slide, and incubated overnight at 4°C. St John's Laboratory Ltd. www.stjohnslabs.com
Figure: Immunohistochemical analysis of paraffin embedded Human liver cancer tissue. 1: GSK3β (phospho Ser9) Polyclonal Antibody was diluted at 1:200 (4 degree Celsius,overnight). 2: Sodium citrate pH 6.0 was used for antibody retrieval (>98 degree Celsius,20min). 3: Secondary antibody was diluted at 1:200 (room temperature, 30min). Negative control was used by secondary antibody only. Report Number 90284-e Host Rabbit Application IHC-P Clonality Polyclonal Model Number STJ90284 Clone ID NA Antibody Name Anti-Phospho-GSK3β (S9) antibody Testing Species HUMAN Testing Tissue LIVER CANCER ANTIBODY VALIDATION REPORT a. (A small amount of distilled water was added into the incubation box to prevent evaporation of antibody). 205. Secondary antibody incubation a. Slides were washed 3 times, with PBS on a shaker for 5min. Shortly after the slides were dried the corresponding secondary antibody solution was added (HRP labelled), covering the tissues, and incubated at room temperature for 30min. b. 206. DAB staining a. Slides were washed 3 times, with PBS on a shaker for 5min. b. Shortly after, the slides were dried and fresh DAB staining buffer was added inside the circles. The staining time was adjusted under a microscope. Yellow-brown colour represented a positive result. Slides were washed with water to stop the staining. c. 207. Haematoxylin staining a. Haematoxylin was used to counter-staining for 1min, and then the slides were washed with water. 1% Hydrochloric acid and alcohol was added for several seconds and then washed with water. Ammonia was used to reveal blue colour, and then flushed with water. b. 208. Desolation and Clearing i. Slides were incubated sequentially into: 75% alcohol 5min, 85% alcohol 5min, Anhydrous ethanol - 5min, Anhydrous ethanol - 5min & Xylene - 5min. Shortly after slides were dried and neutral gum was used to seal the slides. ii. 209. Visualization a. Results were validated with microscope, and the slides were scanned. Paraffin-Embedded Immunohistochemistry Protocol 199. 200. Tissue processing a. Slides were incubated sequentially into Xylene; 15min – Xylene, 15min - Anhydrous ethanol, 5min - Anhydrous ethanol, 5min - 85% alcohol, 5min - 75% alcohol & 5min – wash in distilled water. b. 201. Antigen retrieval a. Tissue slides were incubated with citric acid (PH6.0) antigen retrieval buffer and microwaved for antigen retrieval (heated until boiled and then stopped heating) for 8min. Slides were then heated with medium power for 7min. During this process slides were kept from drying out. After cooling down at room temperature, slides were washed with PBS on shaker for 5min, repeated for 3 times. b. 202. Inhibition of endogenous peroxidase a. Slides were placed in 3% Hydrogen peroxide solution, and incubated for 10 min at room temperature without light exposure. Slides were then washed 3 times with PBS on a shaker for 5mins. b. 203. BSA Blocking a. Shortly after slides were dried, a PAP pen was used to draw circles around the tissue sections (and to prevent draining of the antibody solution). Inside the circles, BSA was used to cover the tissue evenly, blocking for 30min. b. 204. Primary antibody incubation After blocking solution was removed a 1:200 solution of primary antibody/PBS was added on the slide, and incubated overnight at 4°C. St John's Laboratory Ltd. www.stjohnslabs.com
Figure: Immunohistochemical analysis of paraffin embedded Human lung tissue. 1: GSK3β (phospho Ser9) Polyclonal Antibody was diluted at 1:200 (4 degree Celsius,overnight). 2: Sodium citrate pH 6.0 was used for antibody retrieval (>98 degree Celsius,20min). 3: Secondary antibody was diluted at 1:200 (room temperature, 30min). Negative control was used by secondary antibody only. Report Number 90284-f Host Rabbit Application IHC-P Clonality Polyclonal Model Number STJ90284 Clone ID NA Antibody Name Anti-Phospho-GSK3β (S9) antibody Testing Species HUMAN Testing Tissue LUNG ANTIBODY VALIDATION REPORT a. (A small amount of distilled water was added into the incubation box to prevent evaporation of antibody). 194. Secondary antibody incubation a. Slides were washed 3 times, with PBS on a shaker for 5min. Shortly after the slides were dried the corresponding secondary antibody solution was added (HRP labelled), covering the tissues, and incubated at room temperature for 30min. b. 195. DAB staining a. Slides were washed 3 times, with PBS on a shaker for 5min. b. Shortly after, the slides were dried and fresh DAB staining buffer was added inside the circles. The staining time was adjusted under a microscope. Yellow-brown colour represented a positive result. Slides were washed with water to stop the staining. c. 196. Haematoxylin staining a. Haematoxylin was used to counter-staining for 1min, and then the slides were washed with water. 1% Hydrochloric acid and alcohol was added for several seconds and then washed with water. Ammonia was used to reveal blue colour, and then flushed with water. b. 197. Desolation and Clearing i. Slides were incubated sequentially into: 75% alcohol 5min, 85% alcohol 5min, Anhydrous ethanol - 5min, Anhydrous ethanol - 5min & Xylene - 5min. Shortly after slides were dried and neutral gum was used to seal the slides. ii. 198. Visualization a. Results were validated with microscope, and the slides were scanned. Paraffin-Embedded Immunohistochemistry Protocol 188. 189. Tissue processing a. Slides were incubated sequentially into Xylene; 15min – Xylene, 15min - Anhydrous ethanol, 5min - Anhydrous ethanol, 5min - 85% alcohol, 5min - 75% alcohol & 5min – wash in distilled water. b. 190. Antigen retrieval a. Tissue slides were incubated with citric acid (PH6.0) antigen retrieval buffer and microwaved for antigen retrieval (heated until boiled and then stopped heating) for 8min. Slides were then heated with medium power for 7min. During this process slides were kept from drying out. After cooling down at room temperature, slides were washed with PBS on shaker for 5min, repeated for 3 times. b. 191. Inhibition of endogenous peroxidase a. Slides were placed in 3% Hydrogen peroxide solution, and incubated for 10 min at room temperature without light exposure. Slides were then washed 3 times with PBS on a shaker for 5mins. b. 192. BSA Blocking a. Shortly after slides were dried, a PAP pen was used to draw circles around the tissue sections (and to prevent draining of the antibody solution). Inside the circles, BSA was used to cover the tissue evenly, blocking for 30min. b. 193. Primary antibody incubation After blocking solution was removed a 1:200 solution of primary antibody/PBS was added on the slide, and incubated overnight at 4°C. St John's Laboratory Ltd. www.stjohnslabs.com
Figure: Immunohistochemical analysis of paraffin embedded Human stomach tissue. 1: GSK3 β (phospho Ser9) Polyclonal Antibody was diluted at 1:200 (4 degree Celsius,overnight). 2: Sodium citrate pH 6.0 was used for antibody retrieval (>98 degree Celsius,20min). 3: Secondary antibody was diluted at 1:200 (room temperature, 30min). Negative control was used by secondary antibody only. Report Number 90284-g Host Rabbit Application IHC-P Clonality Polyclonal Model Number STJ90284 Clone ID NA Antibody Name Anti-Phospho-GSK3β (S9) antibody Testing Species HUMAN Testing Tissue STOMACH ANTIBODY VALIDATION REPORT a. (A small amount of distilled water was added into the incubation box to prevent evaporation of antibody). 183. Secondary antibody incubation a. Slides were washed 3 times, with PBS on a shaker for 5min. Shortly after the slides were dried the corresponding secondary antibody solution was added (HRP labelled), covering the tissues, and incubated at room temperature for 30min. b. 184. DAB staining a. Slides were washed 3 times, with PBS on a shaker for 5min. b. Shortly after, the slides were dried and fresh DAB staining buffer was added inside the circles. The staining time was adjusted under a microscope. Yellow-brown colour represented a positive result. Slides were washed with water to stop the staining. c. 185. Haematoxylin staining a. Haematoxylin was used to counter-staining for 1min, and then the slides were washed with water. 1% Hydrochloric acid and alcohol was added for several seconds and then washed with water. Ammonia was used to reveal blue colour, and then flushed with water. b. 186. Desolation and Clearing i. Slides were incubated sequentially into: 75% alcohol 5min, 85% alcohol 5min, Anhydrous ethanol - 5min, Anhydrous ethanol - 5min & Xylene - 5min. Shortly after slides were dried and neutral gum was used to seal the slides. ii. 187. Visualization a. Results were validated with microscope, and the slides were scanned. Paraffin-Embedded Immunohistochemistry Protocol 177. 178. Tissue processing a. Slides were incubated sequentially into Xylene; 15min – Xylene, 15min - Anhydrous ethanol, 5min - Anhydrous ethanol, 5min - 85% alcohol, 5min - 75% alcohol & 5min – wash in distilled water. b. 179. Antigen retrieval a. Tissue slides were incubated with citric acid (PH6.0) antigen retrieval buffer and microwaved for antigen retrieval (heated until boiled and then stopped heating) for 8min. Slides were then heated with medium power for 7min. During this process slides were kept from drying out. After cooling down at room temperature, slides were washed with PBS on shaker for 5min, repeated for 3 times. b. 180. Inhibition of endogenous peroxidase a. Slides were placed in 3% Hydrogen peroxide solution, and incubated for 10 min at room temperature without light exposure. Slides were then washed 3 times with PBS on a shaker for 5mins. b. 181. BSA Blocking a. Shortly after slides were dried, a PAP pen was used to draw circles around the tissue sections (and to prevent draining of the antibody solution). Inside the circles, BSA was used to cover the tissue evenly, blocking for 30min. b. 182. Primary antibody incubation After blocking solution was removed a 1:200 solution of primary antibody/PBS was added on the slide, and incubated overnight at 4°C. St John's Laboratory Ltd. www.stjohnslabs.com
Figure: Immunohistochemical analysis of paraffin embedded Human stomach cancer tissue. 1: GSK3β (phospho Ser9) Polyclonal Antibody was diluted at 1:200 (4 degree Celsius,overnight). 2: Sodium citrate pH 6.0 was used for antibody retrieval (>98 degree Celsius,20min). 3: Secondary antibody was diluted at 1:200 (room temperature, 30min). Negative control was used by secondary antibody only. Report Number 90284-h Host Rabbit Application IHC-P Clonality Polyclonal Model Number STJ90284 Clone ID NA Antibody Name Anti-Phospho-GSK3β (S9) antibody Testing Species HUMAN Testing Tissue STOMACH CANCER ANTIBODY VALIDATION REPORT a. (A small amount of distilled water was added into the incubation box to prevent evaporation of antibody). 172. Secondary antibody incubation a. Slides were washed 3 times, with PBS on a shaker for 5min. Shortly after the slides were dried the corresponding secondary antibody solution was added (HRP labelled), covering the tissues, and incubated at room temperature for 30min. b. 173. DAB staining a. Slides were washed 3 times, with PBS on a shaker for 5min. b. Shortly after, the slides were dried and fresh DAB staining buffer was added inside the circles. The staining time was adjusted under a microscope. Yellow-brown colour represented a positive result. Slides were washed with water to stop the staining. c. 174. Haematoxylin staining a. Haematoxylin was used to counter-staining for 1min, and then the slides were washed with water. 1% Hydrochloric acid and alcohol was added for several seconds and then washed with water. Ammonia was used to reveal blue colour, and then flushed with water. b. 175. Desolation and Clearing i. Slides were incubated sequentially into: 75% alcohol 5min, 85% alcohol 5min, Anhydrous ethanol - 5min, Anhydrous ethanol - 5min & Xylene - 5min. Shortly after slides were dried and neutral gum was used to seal the slides. ii. 176. Visualization a. Results were validated with microscope, and the slides were scanned. Paraffin-Embedded Immunohistochemistry Protocol 166. 167. Tissue processing a. Slides were incubated sequentially into Xylene; 15min – Xylene, 15min - Anhydrous ethanol, 5min - Anhydrous ethanol, 5min - 85% alcohol, 5min - 75% alcohol & 5min – wash in distilled water. b. 168. Antigen retrieval a. Tissue slides were incubated with citric acid (PH6.0) antigen retrieval buffer and microwaved for antigen retrieval (heated until boiled and then stopped heating) for 8min. Slides were then heated with medium power for 7min. During this process slides were kept from drying out. After cooling down at room temperature, slides were washed with PBS on shaker for 5min, repeated for 3 times. b. 169. Inhibition of endogenous peroxidase a. Slides were placed in 3% Hydrogen peroxide solution, and incubated for 10 min at room temperature without light exposure. Slides were then washed 3 times with PBS on a shaker for 5mins. b. 170. BSA Blocking a. Shortly after slides were dried, a PAP pen was used to draw circles around the tissue sections (and to prevent draining of the antibody solution). Inside the circles, BSA was used to cover the tissue evenly, blocking for 30min. b. 171. Primary antibody incubation After blocking solution was removed a 1:200 solution of primary antibody/PBS was added on the slide, and incubated overnight at 4°C. St John's Laboratory Ltd. www.stjohnslabs.com
Figure: Immunohistochemical analysis of paraffin embedded Human appendix tissue. 1: GSK3β (phospho Ser9) Polyclonal Antibody was diluted at 1:200 (4 degree Celsius,overnight). 2: Sodium citrate pH 6.0 was used for antibody retrieval (>98 degree Celsius,20min). 3: Secondary antibody was diluted at 1:200 (room temperature, 30min). Negative control was used by secondary antibody only. Report Number 90284-i Host Rabbit Application IHC-P Clonality Polyclonal Model Number STJ90284 Clone ID NA Antibody Name Anti-Phospho-GSK3β (S9) antibody Testing Species HUMAN Testing Tissue APPENDIX ANTIBODY VALIDATION REPORT a. (A small amount of distilled water was added into the incubation box to prevent evaporation of antibody). 161. Secondary antibody incubation a. Slides were washed 3 times, with PBS on a shaker for 5min. Shortly after the slides were dried the corresponding secondary antibody solution was added (HRP labelled), covering the tissues, and incubated at room temperature for 30min. b. 162. DAB staining a. Slides were washed 3 times, with PBS on a shaker for 5min. b. Shortly after, the slides were dried and fresh DAB staining buffer was added inside the circles. The staining time was adjusted under a microscope. Yellow-brown colour represented a positive result. Slides were washed with water to stop the staining. c. 163. Haematoxylin staining a. Haematoxylin was used to counter-staining for 1min, and then the slides were washed with water. 1% Hydrochloric acid and alcohol was added for several seconds and then washed with water. Ammonia was used to reveal blue colour, and then flushed with water. b. 164. Desolation and Clearing i. Slides were incubated sequentially into: 75% alcohol 5min, 85% alcohol 5min, Anhydrous ethanol - 5min, Anhydrous ethanol - 5min & Xylene - 5min. Shortly after slides were dried and neutral gum was used to seal the slides. ii. 165. Visualization a. Results were validated with microscope, and the slides were scanned. Paraffin-Embedded Immunohistochemistry Protocol 155. 156. Tissue processing a. Slides were incubated sequentially into Xylene; 15min – Xylene, 15min - Anhydrous ethanol, 5min - Anhydrous ethanol, 5min - 85% alcohol, 5min - 75% alcohol & 5min – wash in distilled water. b. 157. Antigen retrieval a. Tissue slides were incubated with citric acid (PH6.0) antigen retrieval buffer and microwaved for antigen retrieval (heated until boiled and then stopped heating) for 8min. Slides were then heated with medium power for 7min. During this process slides were kept from drying out. After cooling down at room temperature, slides were washed with PBS on shaker for 5min, repeated for 3 times. b. 158. Inhibition of endogenous peroxidase a. Slides were placed in 3% Hydrogen peroxide solution, and incubated for 10 min at room temperature without light exposure. Slides were then washed 3 times with PBS on a shaker for 5mins. b. 159. BSA Blocking a. Shortly after slides were dried, a PAP pen was used to draw circles around the tissue sections (and to prevent draining of the antibody solution). Inside the circles, BSA was used to cover the tissue evenly, blocking for 30min. b. 160. Primary antibody incubation After blocking solution was removed a 1:200 solution of primary antibody/PBS was added on the slide, and incubated overnight at 4°C. St John's Laboratory Ltd. www.stjohnslabs.com
Figure: Immunohistochemical analysis of paraffin embedded Rat heart tissue. 1: GSK3β (phospho Ser9) Polyclonal Antibody was diluted at 1:200 (4 degree Celsius,overnight). 2: Sodium citrate pH 6.0 was used for antibody retrieval (>98 degree Celsius,20min). 3: Secondary antibody was diluted at 1:200 (room temperature, 30min). Negative control was used by secondary antibody only. Report Number 90284-j Host Rabbit Application IHC-P Clonality Polyclonal Model Number STJ90284 Clone ID NA Antibody Name Anti-Phospho-GSK3β (S9) antibody Testing Species RAT Testing Tissue HEART ANTIBODY VALIDATION REPORT a. (A small amount of distilled water was added into the incubation box to prevent evaporation of antibody). 150. Secondary antibody incubation a. Slides were washed 3 times, with PBS on a shaker for 5min. Shortly after the slides were dried the corresponding secondary antibody solution was added (HRP labelled), covering the tissues, and incubated at room temperature for 30min. b. 151. DAB staining a. Slides were washed 3 times, with PBS on a shaker for 5min. b. Shortly after, the slides were dried and fresh DAB staining buffer was added inside the circles. The staining time was adjusted under a microscope. Yellow-brown colour represented a positive result. Slides were washed with water to stop the staining. c. 152. Haematoxylin staining a. Haematoxylin was used to counter-staining for 1min, and then the slides were washed with water. 1% Hydrochloric acid and alcohol was added for several seconds and then washed with water. Ammonia was used to reveal blue colour, and then flushed with water. b. 153. Desolation and Clearing i. Slides were incubated sequentially into: 75% alcohol 5min, 85% alcohol 5min, Anhydrous ethanol - 5min, Anhydrous ethanol - 5min & Xylene - 5min. Shortly after slides were dried and neutral gum was used to seal the slides. ii. 154. Visualization a. Results were validated with microscope, and the slides were scanned. Paraffin-Embedded Immunohistochemistry Protocol 144. 145. Tissue processing a. Slides were incubated sequentially into Xylene; 15min – Xylene, 15min - Anhydrous ethanol, 5min - Anhydrous ethanol, 5min - 85% alcohol, 5min - 75% alcohol & 5min – wash in distilled water. b. 146. Antigen retrieval a. Tissue slides were incubated with citric acid (PH6.0) antigen retrieval buffer and microwaved for antigen retrieval (heated until boiled and then stopped heating) for 8min. Slides were then heated with medium power for 7min. During this process slides were kept from drying out. After cooling down at room temperature, slides were washed with PBS on shaker for 5min, repeated for 3 times. b. 147. Inhibition of endogenous peroxidase a. Slides were placed in 3% Hydrogen peroxide solution, and incubated for 10 min at room temperature without light exposure. Slides were then washed 3 times with PBS on a shaker for 5mins. b. 148. BSA Blocking a. Shortly after slides were dried, a PAP pen was used to draw circles around the tissue sections (and to prevent draining of the antibody solution). Inside the circles, BSA was used to cover the tissue evenly, blocking for 30min. b. 149. Primary antibody incubation After blocking solution was removed a 1:200 solution of primary antibody/PBS was added on the slide, and incubated overnight at 4°C. St John's Laboratory Ltd. www.stjohnslabs.com
Figure: Immunohistochemical analysis of paraffin embedded Rat testis tissue. 1: GSK3β (phospho Ser9) Polyclonal Antibody was diluted at 1:200 (4 degree Celsius,overnight). 2: Sodium citrate pH 6.0 was used for antibody retrieval (>98 degree Celsius,20min). 3: Secondary antibody was diluted at 1:200 (room temperature, 30min). Negative control was used by secondary antibody only. Report Number 90284-k Host Rabbit Application IHC-P Clonality Polyclonal Model Number STJ90284 Clone ID NA Antibody Name Anti-Phospho-GSK3β (S9) antibody Testing Species RAT Testing Tissue TESTIS ANTIBODY VALIDATION REPORT a. (A small amount of distilled water was added into the incubation box to prevent evaporation of antibody). 139. Secondary antibody incubation a. Slides were washed 3 times, with PBS on a shaker for 5min. Shortly after the slides were dried the corresponding secondary antibody solution was added (HRP labelled), covering the tissues, and incubated at room temperature for 30min. b. 140. DAB staining a. Slides were washed 3 times, with PBS on a shaker for 5min. b. Shortly after, the slides were dried and fresh DAB staining buffer was added inside the circles. The staining time was adjusted under a microscope. Yellow-brown colour represented a positive result. Slides were washed with water to stop the staining. c. 141. Haematoxylin staining a. Haematoxylin was used to counter-staining for 1min, and then the slides were washed with water. 1% Hydrochloric acid and alcohol was added for several seconds and then washed with water. Ammonia was used to reveal blue colour, and then flushed with water. b. 142. Desolation and Clearing i. Slides were incubated sequentially into: 75% alcohol 5min, 85% alcohol 5min, Anhydrous ethanol - 5min, Anhydrous ethanol - 5min & Xylene - 5min. Shortly after slides were dried and neutral gum was used to seal the slides. ii. 143. Visualization a. Results were validated with microscope, and the slides were scanned. Paraffin-Embedded Immunohistochemistry Protocol 133. 134. Tissue processing a. Slides were incubated sequentially into Xylene; 15min – Xylene, 15min - Anhydrous ethanol, 5min - Anhydrous ethanol, 5min - 85% alcohol, 5min - 75% alcohol & 5min – wash in distilled water. b. 135. Antigen retrieval a. Tissue slides were incubated with citric acid (PH6.0) antigen retrieval buffer and microwaved for antigen retrieval (heated until boiled and then stopped heating) for 8min. Slides were then heated with medium power for 7min. During this process slides were kept from drying out. After cooling down at room temperature, slides were washed with PBS on shaker for 5min, repeated for 3 times. b. 136. Inhibition of endogenous peroxidase a. Slides were placed in 3% Hydrogen peroxide solution, and incubated for 10 min at room temperature without light exposure. Slides were then washed 3 times with PBS on a shaker for 5mins. b. 137. BSA Blocking a. Shortly after slides were dried, a PAP pen was used to draw circles around the tissue sections (and to prevent draining of the antibody solution). Inside the circles, BSA was used to cover the tissue evenly, blocking for 30min. b. 138. Primary antibody incubation After blocking solution was removed a 1:200 solution of primary antibody/PBS was added on the slide, and incubated overnight at 4°C. St John's Laboratory Ltd. www.stjohnslabs.com
Figure: Immunohistochemical analysis of paraffin embedded Rat liver tissue. 1: GSK3β (phospho Ser9) Polyclonal Antibody was diluted at 1:200 (4 degree Celsius,overnight). 2: Sodium citrate pH 6.0 was used for antibody retrieval (>98 degree Celsius,20min). 3: Secondary antibody was diluted at 1:200 (room temperature, 30min). Negative control was used by secondary antibody only. Report Number 90284-l Host Rabbit Application IHC-P Clonality Polyclonal Model Number STJ90284 Clone ID NA Antibody Name Anti-Phospho-GSK3β (S9) antibody Testing Species RAT Testing Tissue LIVER ANTIBODY VALIDATION REPORT a. (A small amount of distilled water was added into the incubation box to prevent evaporation of antibody). 128. Secondary antibody incubation a. Slides were washed 3 times, with PBS on a shaker for 5min. Shortly after the slides were dried the corresponding secondary antibody solution was added (HRP labelled), covering the tissues, and incubated at room temperature for 30min. b. 129. DAB staining a. Slides were washed 3 times, with PBS on a shaker for 5min. b. Shortly after, the slides were dried and fresh DAB staining buffer was added inside the circles. The staining time was adjusted under a microscope. Yellow-brown colour represented a positive result. Slides were washed with water to stop the staining. c. 130. Haematoxylin staining a. Haematoxylin was used to counter-staining for 1min, and then the slides were washed with water. 1% Hydrochloric acid and alcohol was added for several seconds and then washed with water. Ammonia was used to reveal blue colour, and then flushed with water. b. 131. Desolation and Clearing i. Slides were incubated sequentially into: 75% alcohol 5min, 85% alcohol 5min, Anhydrous ethanol - 5min, Anhydrous ethanol - 5min & Xylene - 5min. Shortly after slides were dried and neutral gum was used to seal the slides. ii. 132. Visualization a. Results were validated with microscope, and the slides were scanned. Paraffin-Embedded Immunohistochemistry Protocol 122. 123. Tissue processing a. Slides were incubated sequentially into Xylene; 15min – Xylene, 15min - Anhydrous ethanol, 5min - Anhydrous ethanol, 5min - 85% alcohol, 5min - 75% alcohol & 5min – wash in distilled water. b. 124. Antigen retrieval a. Tissue slides were incubated with citric acid (PH6.0) antigen retrieval buffer and microwaved for antigen retrieval (heated until boiled and then stopped heating) for 8min. Slides were then heated with medium power for 7min. During this process slides were kept from drying out. After cooling down at room temperature, slides were washed with PBS on shaker for 5min, repeated for 3 times. b. 125. Inhibition of endogenous peroxidase a. Slides were placed in 3% Hydrogen peroxide solution, and incubated for 10 min at room temperature without light exposure. Slides were then washed 3 times with PBS on a shaker for 5mins. b. 126. BSA Blocking a. Shortly after slides were dried, a PAP pen was used to draw circles around the tissue sections (and to prevent draining of the antibody solution). Inside the circles, BSA was used to cover the tissue evenly, blocking for 30min. b. 127. Primary antibody incubation After blocking solution was removed a 1:200 solution of primary antibody/PBS was added on the slide, and incubated overnight at 4°C. St John's Laboratory Ltd. www.stjohnslabs.com
Figure: Immunohistochemical analysis of paraffin embedded Rat lung tissue. 1: GSK3β (phospho Ser9) Polyclonal Antibody was diluted at 1:200 (4 degree Celsius,overnight). 2: Sodium citrate pH 6.0 was used for antibody retrieval (>98 degree Celsius,20min). 3: Secondary antibody was diluted at 1:200 (room temperature, 30min). Negative control was used by secondary antibody only. Report Number 90284-m Host Rabbit Application IHC-P Clonality Polyclonal Model Number STJ90284 Clone ID NA Antibody Name Anti-Phospho-GSK3β (S9) antibody Testing Species RAT Testing Tissue LUNG ANTIBODY VALIDATION REPORT a. (A small amount of distilled water was added into the incubation box to prevent evaporation of antibody). 117. Secondary antibody incubation a. Slides were washed 3 times, with PBS on a shaker for 5min. Shortly after the slides were dried the corresponding secondary antibody solution was added (HRP labelled), covering the tissues, and incubated at room temperature for 30min. b. 118. DAB staining a. Slides were washed 3 times, with PBS on a shaker for 5min. b. Shortly after, the slides were dried and fresh DAB staining buffer was added inside the circles. The staining time was adjusted under a microscope. Yellow-brown colour represented a positive result. Slides were washed with water to stop the staining. c. 119. Haematoxylin staining a. Haematoxylin was used to counter-staining for 1min, and then the slides were washed with water. 1% Hydrochloric acid and alcohol was added for several seconds and then washed with water. Ammonia was used to reveal blue colour, and then flushed with water. b. 120. Desolation and Clearing i. Slides were incubated sequentially into: 75% alcohol 5min, 85% alcohol 5min, Anhydrous ethanol - 5min, Anhydrous ethanol - 5min & Xylene - 5min. Shortly after slides were dried and neutral gum was used to seal the slides. ii. 121. Visualization a. Results were validated with microscope, and the slides were scanned. Paraffin-Embedded Immunohistochemistry Protocol 111. 112. Tissue processing a. Slides were incubated sequentially into Xylene; 15min – Xylene, 15min - Anhydrous ethanol, 5min - Anhydrous ethanol, 5min - 85% alcohol, 5min - 75% alcohol & 5min – wash in distilled water. b. 113. Antigen retrieval a. Tissue slides were incubated with citric acid (PH6.0) antigen retrieval buffer and microwaved for antigen retrieval (heated until boiled and then stopped heating) for 8min. Slides were then heated with medium power for 7min. During this process slides were kept from drying out. After cooling down at room temperature, slides were washed with PBS on shaker for 5min, repeated for 3 times. b. 114. Inhibition of endogenous peroxidase a. Slides were placed in 3% Hydrogen peroxide solution, and incubated for 10 min at room temperature without light exposure. Slides were then washed 3 times with PBS on a shaker for 5mins. b. 115. BSA Blocking a. Shortly after slides were dried, a PAP pen was used to draw circles around the tissue sections (and to prevent draining of the antibody solution). Inside the circles, BSA was used to cover the tissue evenly, blocking for 30min. b. 116. Primary antibody incubation After blocking solution was removed a 1:200 solution of primary antibody/PBS was added on the slide, and incubated overnight at 4°C. St John's Laboratory Ltd. www.stjohnslabs.com
Figure: Immunohistochemical analysis of paraffin embedded Rat kidney tissue. 1: GSK3β (phospho Ser9) Polyclonal Antibody was diluted at 1:200 (4 degree Celsius,overnight). 2: Sodium citrate pH 6.0 was used for antibody retrieval (>98 degree Celsius,20min). 3: Secondary antibody was diluted at 1:200 (room temperature, 30min). Negative control was used by secondary antibody only. Report Number 90284-n Host Rabbit Application IHC-P Clonality Polyclonal Model Number STJ90284 Clone ID NA Antibody Name Anti-Phospho-GSK3β (S9) antibody Testing Species RAT Testing Tissue KIDNEY ANTIBODY VALIDATION REPORT a. (A small amount of distilled water was added into the incubation box to prevent evaporation of antibody). 106. Secondary antibody incubation a. Slides were washed 3 times, with PBS on a shaker for 5min. Shortly after the slides were dried the corresponding secondary antibody solution was added (HRP labelled), covering the tissues, and incubated at room temperature for 30min. b. 107. DAB staining a. Slides were washed 3 times, with PBS on a shaker for 5min. b. Shortly after, the slides were dried and fresh DAB staining buffer was added inside the circles. The staining time was adjusted under a microscope. Yellow-brown colour represented a positive result. Slides were washed with water to stop the staining. c. 108. Haematoxylin staining a. Haematoxylin was used to counter-staining for 1min, and then the slides were washed with water. 1% Hydrochloric acid and alcohol was added for several seconds and then washed with water. Ammonia was used to reveal blue colour, and then flushed with water. b. 109. Desolation and Clearing i. Slides were incubated sequentially into: 75% alcohol 5min, 85% alcohol 5min, Anhydrous ethanol - 5min, Anhydrous ethanol - 5min & Xylene - 5min. Shortly after slides were dried and neutral gum was used to seal the slides. ii. 110. Visualization a. Results were validated with microscope, and the slides were scanned. Paraffin-Embedded Immunohistochemistry Protocol 100. 101. Tissue processing a. Slides were incubated sequentially into Xylene; 15min – Xylene, 15min - Anhydrous ethanol, 5min - Anhydrous ethanol, 5min - 85% alcohol, 5min - 75% alcohol & 5min – wash in distilled water. b. 102. Antigen retrieval a. Tissue slides were incubated with citric acid (PH6.0) antigen retrieval buffer and microwaved for antigen retrieval (heated until boiled and then stopped heating) for 8min. Slides were then heated with medium power for 7min. During this process slides were kept from drying out. After cooling down at room temperature, slides were washed with PBS on shaker for 5min, repeated for 3 times. b. 103. Inhibition of endogenous peroxidase a. Slides were placed in 3% Hydrogen peroxide solution, and incubated for 10 min at room temperature without light exposure. Slides were then washed 3 times with PBS on a shaker for 5mins. b. 104. BSA Blocking a. Shortly after slides were dried, a PAP pen was used to draw circles around the tissue sections (and to prevent draining of the antibody solution). Inside the circles, BSA was used to cover the tissue evenly, blocking for 30min. b. 105. Primary antibody incubation After blocking solution was removed a 1:200 solution of primary antibody/PBS was added on the slide, and incubated overnight at 4°C. St John's Laboratory Ltd. www.stjohnslabs.com
Figure: Immunohistochemical analysis of paraffin embedded Rat spinal cord tissue. 1: GSK3β (phospho Ser9) Polyclonal Antibody was diluted at 1:200 (4 degree Celsius,overnight). 2: Sodium citrate pH 6.0 was used for antibody retrieval (>98 degree Celsius,20min). 3: Secondary antibody was diluted at 1:200 (room temperature, 30min). Negative control was used by secondary antibody only. Report Number 90284-o Host Rabbit Application IHC-P Clonality Polyclonal Model Number STJ90284 Clone ID NA Antibody Name Anti-Phospho-GSK3β (S9) antibody Testing Species RAT Testing Tissue SPINAL ANTIBODY VALIDATION REPORT a. (A small amount of distilled water was added into the incubation box to prevent evaporation of antibody). 95. Secondary antibody incubation a. Slides were washed 3 times, with PBS on a shaker for 5min. Shortly after the slides were dried the corresponding secondary antibody solution was added (HRP labelled), covering the tissues, and incubated at room temperature for 30min. b. 96. DAB staining a. Slides were washed 3 times, with PBS on a shaker for 5min. b. Shortly after, the slides were dried and fresh DAB staining buffer was added inside the circles. The staining time was adjusted under a microscope. Yellow-brown colour represented a positive result. Slides were washed with water to stop the staining. c. 97. Haematoxylin staining a. Haematoxylin was used to counter-staining for 1min, and then the slides were washed with water. 1% Hydrochloric acid and alcohol was added for several seconds and then washed with water. Ammonia was used to reveal blue colour, and then flushed with water. b. 98. Desolation and Clearing i. Slides were incubated sequentially into: 75% alcohol 5min, 85% alcohol 5min, Anhydrous ethanol - 5min, Anhydrous ethanol - 5min & Xylene - 5min. Shortly after slides were dried and neutral gum was used to seal the slides. ii. 99. Visualization a. Results were validated with microscope, and the slides were scanned. Paraffin-Embedded Immunohistochemistry Protocol 89. 90. Tissue processing a. Slides were incubated sequentially into Xylene; 15min – Xylene, 15min - Anhydrous ethanol, 5min - Anhydrous ethanol, 5min - 85% alcohol, 5min - 75% alcohol & 5min – wash in distilled water. b. 91. Antigen retrieval a. Tissue slides were incubated with citric acid (PH6.0) antigen retrieval buffer and microwaved for antigen retrieval (heated until boiled and then stopped heating) for 8min. Slides were then heated with medium power for 7min. During this process slides were kept from drying out. After cooling down at room temperature, slides were washed with PBS on shaker for 5min, repeated for 3 times. b. 92. Inhibition of endogenous peroxidase a. Slides were placed in 3% Hydrogen peroxide solution, and incubated for 10 min at room temperature without light exposure. Slides were then washed 3 times with PBS on a shaker for 5mins. b. 93. BSA Blocking a. Shortly after slides were dried, a PAP pen was used to draw circles around the tissue sections (and to prevent draining of the antibody solution). Inside the circles, BSA was used to cover the tissue evenly, blocking for 30min. b. 94. Primary antibody incubation After blocking solution was removed a 1:200 solution of primary antibody/PBS was added on the slide, and incubated overnight at 4°C. St John's Laboratory Ltd. www.stjohnslabs.com
Figure: Immunohistochemical analysis of paraffin embedded Rat brain tissue. 1: GSK3β (phospho Ser9) Polyclonal Antibody was diluted at 1:200 (4 degree Celsius,overnight). 2: Sodium citrate pH 6.0 was used for antibody retrieval (>98 degree Celsius,20min). 3: Secondary antibody was diluted at 1:200 (room temperature, 30min). Negative control was used by secondary antibody only. Report Number 90284-p Host Rabbit Application IHC-P Clonality Polyclonal Model Number STJ90284 Clone ID NA Antibody Name Anti-Phospho-GSK3β (S9) antibody Testing Species RAT Testing Tissue BRAIN ANTIBODY VALIDATION REPORT a. (A small amount of distilled water was added into the incubation box to prevent evaporation of antibody). 84. Secondary antibody incubation a. Slides were washed 3 times, with PBS on a shaker for 5min. Shortly after the slides were dried the corresponding secondary antibody solution was added (HRP labelled), covering the tissues, and incubated at room temperature for 30min. b. 85. DAB staining a. Slides were washed 3 times, with PBS on a shaker for 5min. b. Shortly after, the slides were dried and fresh DAB staining buffer was added inside the circles. The staining time was adjusted under a microscope. Yellow-brown colour represented a positive result. Slides were washed with water to stop the staining. c. 86. Haematoxylin staining a. Haematoxylin was used to counter-staining for 1min, and then the slides were washed with water. 1% Hydrochloric acid and alcohol was added for several seconds and then washed with water. Ammonia was used to reveal blue colour, and then flushed with water. b. 87. Desolation and Clearing i. Slides were incubated sequentially into: 75% alcohol 5min, 85% alcohol 5min, Anhydrous ethanol - 5min, Anhydrous ethanol - 5min & Xylene - 5min. Shortly after slides were dried and neutral gum was used to seal the slides. ii. 88. Visualization a. Results were validated with microscope, and the slides were scanned. Paraffin-Embedded Immunohistochemistry Protocol 78. 79. Tissue processing a. Slides were incubated sequentially into Xylene; 15min – Xylene, 15min - Anhydrous ethanol, 5min - Anhydrous ethanol, 5min - 85% alcohol, 5min - 75% alcohol & 5min – wash in distilled water. b. 80. Antigen retrieval a. Tissue slides were incubated with citric acid (PH6.0) antigen retrieval buffer and microwaved for antigen retrieval (heated until boiled and then stopped heating) for 8min. Slides were then heated with medium power for 7min. During this process slides were kept from drying out. After cooling down at room temperature, slides were washed with PBS on shaker for 5min, repeated for 3 times. b. 81. Inhibition of endogenous peroxidase a. Slides were placed in 3% Hydrogen peroxide solution, and incubated for 10 min at room temperature without light exposure. Slides were then washed 3 times with PBS on a shaker for 5mins. b. 82. BSA Blocking a. Shortly after slides were dried, a PAP pen was used to draw circles around the tissue sections (and to prevent draining of the antibody solution). Inside the circles, BSA was used to cover the tissue evenly, blocking for 30min. b. 83. Primary antibody incubation After blocking solution was removed a 1:200 solution of primary antibody/PBS was added on the slide, and incubated overnight at 4°C. St John's Laboratory Ltd. www.stjohnslabs.com
Figure: Immunohistochemical analysis of paraffin embedded Rat spleen tissue. 1: GSK3β (phospho Ser9) Polyclonal Antibody was diluted at 1:200 (4 degree Celsius,overnight). 2: Sodium citrate pH 6.0 was used for antibody retrieval (>98 degree Celsius,20min). 3: Secondary antibody was diluted at 1:200 (room temperature, 30min). Negative control was used by secondary antibody only. Report Number 90284-q Host Rabbit Application IHC-P Clonality Polyclonal Model Number STJ90284 Clone ID NA Antibody Name Anti-Phospho-GSK3β (S9) antibody Testing Species RAT Testing Tissue SPLEEN ANTIBODY VALIDATION REPORT a. (A small amount of distilled water was added into the incubation box to prevent evaporation of antibody). 73. Secondary antibody incubation a. Slides were washed 3 times, with PBS on a shaker for 5min. Shortly after the slides were dried the corresponding secondary antibody solution was added (HRP labelled), covering the tissues, and incubated at room temperature for 30min. b. 74. DAB staining a. Slides were washed 3 times, with PBS on a shaker for 5min. b. Shortly after, the slides were dried and fresh DAB staining buffer was added inside the circles. The staining time was adjusted under a microscope. Yellow-brown colour represented a positive result. Slides were washed with water to stop the staining. c. 75. Haematoxylin staining a. Haematoxylin was used to counter-staining for 1min, and then the slides were washed with water. 1% Hydrochloric acid and alcohol was added for several seconds and then washed with water. Ammonia was used to reveal blue colour, and then flushed with water. b. 76. Desolation and Clearing i. Slides were incubated sequentially into: 75% alcohol 5min, 85% alcohol 5min, Anhydrous ethanol - 5min, Anhydrous ethanol - 5min & Xylene - 5min. Shortly after slides were dried and neutral gum was used to seal the slides. ii. 77. Visualization a. Results were validated with microscope, and the slides were scanned. Paraffin-Embedded Immunohistochemistry Protocol 67. 68. Tissue processing a. Slides were incubated sequentially into Xylene; 15min – Xylene, 15min - Anhydrous ethanol, 5min - Anhydrous ethanol, 5min - 85% alcohol, 5min - 75% alcohol & 5min – wash in distilled water. b. 69. Antigen retrieval a. Tissue slides were incubated with citric acid (PH6.0) antigen retrieval buffer and microwaved for antigen retrieval (heated until boiled and then stopped heating) for 8min. Slides were then heated with medium power for 7min. During this process slides were kept from drying out. After cooling down at room temperature, slides were washed with PBS on shaker for 5min, repeated for 3 times. b. 70. Inhibition of endogenous peroxidase a. Slides were placed in 3% Hydrogen peroxide solution, and incubated for 10 min at room temperature without light exposure. Slides were then washed 3 times with PBS on a shaker for 5mins. b. 71. BSA Blocking a. Shortly after slides were dried, a PAP pen was used to draw circles around the tissue sections (and to prevent draining of the antibody solution). Inside the circles, BSA was used to cover the tissue evenly, blocking for 30min. b. 72. Primary antibody incubation After blocking solution was removed a 1:200 solution of primary antibody/PBS was added on the slide, and incubated overnight at 4°C. St John's Laboratory Ltd. www.stjohnslabs.com
Figure: Immunohistochemical analysis of paraffin embedded Mouse heart tissue. 1: GSK3β (phospho Ser9) Polyclonal Antibody was diluted at 1:200 (4 degree Celsius,overnight). 2: Sodium citrate pH 6.0 was used for antibody retrieval (>98 degree Celsius,20min). 3: Secondary antibody was diluted at 1:200 (room temperature, 30min). Negative control was used by secondary antibody only. Report Number 90284-r Host Rabbit Application IHC-P Clonality Polyclonal Model Number STJ90284 Clone ID NA Antibody Name Anti-Phospho-GSK3β (S9) antibody Testing Species MOUSE Testing Tissue HEART ANTIBODY VALIDATION REPORT a. (A small amount of distilled water was added into the incubation box to prevent evaporation of antibody). 62. Secondary antibody incubation a. Slides were washed 3 times, with PBS on a shaker for 5min. Shortly after the slides were dried the corresponding secondary antibody solution was added (HRP labelled), covering the tissues, and incubated at room temperature for 30min. b. 63. DAB staining a. Slides were washed 3 times, with PBS on a shaker for 5min. b. Shortly after, the slides were dried and fresh DAB staining buffer was added inside the circles. The staining time was adjusted under a microscope. Yellow-brown colour represented a positive result. Slides were washed with water to stop the staining. c. 64. Haematoxylin staining a. Haematoxylin was used to counter-staining for 1min, and then the slides were washed with water. 1% Hydrochloric acid and alcohol was added for several seconds and then washed with water. Ammonia was used to reveal blue colour, and then flushed with water. b. 65. Desolation and Clearing i. Slides were incubated sequentially into: 75% alcohol 5min, 85% alcohol 5min, Anhydrous ethanol - 5min, Anhydrous ethanol - 5min & Xylene - 5min. Shortly after slides were dried and neutral gum was used to seal the slides. ii. 66. Visualization a. Results were validated with microscope, and the slides were scanned. Paraffin-Embedded Immunohistochemistry Protocol 56. 57. Tissue processing a. Slides were incubated sequentially into Xylene; 15min – Xylene, 15min - Anhydrous ethanol, 5min - Anhydrous ethanol, 5min - 85% alcohol, 5min - 75% alcohol & 5min – wash in distilled water. b. 58. Antigen retrieval a. Tissue slides were incubated with citric acid (PH6.0) antigen retrieval buffer and microwaved for antigen retrieval (heated until boiled and then stopped heating) for 8min. Slides were then heated with medium power for 7min. During this process slides were kept from drying out. After cooling down at room temperature, slides were washed with PBS on shaker for 5min, repeated for 3 times. b. 59. Inhibition of endogenous peroxidase a. Slides were placed in 3% Hydrogen peroxide solution, and incubated for 10 min at room temperature without light exposure. Slides were then washed 3 times with PBS on a shaker for 5mins. b. 60. BSA Blocking a. Shortly after slides were dried, a PAP pen was used to draw circles around the tissue sections (and to prevent draining of the antibody solution). Inside the circles, BSA was used to cover the tissue evenly, blocking for 30min. b. 61. Primary antibody incubation After blocking solution was removed a 1:200 solution of primary antibody/PBS was added on the slide, and incubated overnight at 4°C. St John's Laboratory Ltd. www.stjohnslabs.com
Figure: Immunohistochemical analysis of paraffin embedded Mouse testis tissue. 1: GSK3β (phospho Ser9) Polyclonal Antibody was diluted at 1:200 (4 degree Celsius,overnight). 2: Sodium citrate pH 6.0 was used for antibody retrieval (>98 degree Celsius,20min). 3: Secondary antibody was diluted at 1:200 (room temperature, 30min). Negative control was used by secondary antibody only. Report Number 90284-s Host Rabbit Application IHC-P Clonality Polyclonal Model Number STJ90284 Clone ID NA Antibody Name Anti-Phospho-GSK3β (S9) antibody Testing Species MOUSE Testing Tissue TESTIS ANTIBODY VALIDATION REPORT a. (A small amount of distilled water was added into the incubation box to prevent evaporation of antibody). 51. Secondary antibody incubation a. Slides were washed 3 times, with PBS on a shaker for 5min. Shortly after the slides were dried the corresponding secondary antibody solution was added (HRP labelled), covering the tissues, and incubated at room temperature for 30min. b. 52. DAB staining a. Slides were washed 3 times, with PBS on a shaker for 5min. b. Shortly after, the slides were dried and fresh DAB staining buffer was added inside the circles. The staining time was adjusted under a microscope. Yellow-brown colour represented a positive result. Slides were washed with water to stop the staining. c. 53. Haematoxylin staining a. Haematoxylin was used to counter-staining for 1min, and then the slides were washed with water. 1% Hydrochloric acid and alcohol was added for several seconds and then washed with water. Ammonia was used to reveal blue colour, and then flushed with water. b. 54. Desolation and Clearing i. Slides were incubated sequentially into: 75% alcohol 5min, 85% alcohol 5min, Anhydrous ethanol - 5min, Anhydrous ethanol - 5min & Xylene - 5min. Shortly after slides were dried and neutral gum was used to seal the slides. ii. 55. Visualization a. Results were validated with microscope, and the slides were scanned. Paraffin-Embedded Immunohistochemistry Protocol 45. 46. Tissue processing a. Slides were incubated sequentially into Xylene; 15min – Xylene, 15min - Anhydrous ethanol, 5min - Anhydrous ethanol, 5min - 85% alcohol, 5min - 75% alcohol & 5min – wash in distilled water. b. 47. Antigen retrieval a. Tissue slides were incubated with citric acid (PH6.0) antigen retrieval buffer and microwaved for antigen retrieval (heated until boiled and then stopped heating) for 8min. Slides were then heated with medium power for 7min. During this process slides were kept from drying out. After cooling down at room temperature, slides were washed with PBS on shaker for 5min, repeated for 3 times. b. 48. Inhibition of endogenous peroxidase a. Slides were placed in 3% Hydrogen peroxide solution, and incubated for 10 min at room temperature without light exposure. Slides were then washed 3 times with PBS on a shaker for 5mins. b. 49. BSA Blocking a. Shortly after slides were dried, a PAP pen was used to draw circles around the tissue sections (and to prevent draining of the antibody solution). Inside the circles, BSA was used to cover the tissue evenly, blocking for 30min. b. 50. Primary antibody incubation After blocking solution was removed a 1:200 solution of primary antibody/PBS was added on the slide, and incubated overnight at 4°C. St John's Laboratory Ltd. www.stjohnslabs.com
Figure: Immunohistochemical analysis of paraffin embedded Mouse liver tissue. 1: GSK3β (phospho Ser9) Polyclonal Antibody was diluted at 1:200 (4 degree Celsius,overnight). 2: Sodium citrate pH 6.0 was used for antibody retrieval (>98 degree Celsius,20min). 3: Secondary antibody was diluted at 1:200 (room temperature, 30min). Negative control was used by secondary antibody only. Report Number 90284-t Host Rabbit Application IHC-P Clonality Polyclonal Model Number STJ90284 Clone ID NA Antibody Name Anti-Phospho-GSK3β (S9) antibody Testing Species MOUSE Testing Tissue LIVER ANTIBODY VALIDATION REPORT a. (A small amount of distilled water was added into the incubation box to prevent evaporation of antibody). 40. Secondary antibody incubation a. Slides were washed 3 times, with PBS on a shaker for 5min. Shortly after the slides were dried the corresponding secondary antibody solution was added (HRP labelled), covering the tissues, and incubated at room temperature for 30min. b. 41. DAB staining a. Slides were washed 3 times, with PBS on a shaker for 5min. b. Shortly after, the slides were dried and fresh DAB staining buffer was added inside the circles. The staining time was adjusted under a microscope. Yellow-brown colour represented a positive result. Slides were washed with water to stop the staining. c. 42. Haematoxylin staining a. Haematoxylin was used to counter-staining for 1min, and then the slides were washed with water. 1% Hydrochloric acid and alcohol was added for several seconds and then washed with water. Ammonia was used to reveal blue colour, and then flushed with water. b. 43. Desolation and Clearing i. Slides were incubated sequentially into: 75% alcohol 5min, 85% alcohol 5min, Anhydrous ethanol - 5min, Anhydrous ethanol - 5min & Xylene - 5min. Shortly after slides were dried and neutral gum was used to seal the slides. ii. 44. Visualization a. Results were validated with microscope, and the slides were scanned. Paraffin-Embedded Immunohistochemistry Protocol 34. 35. Tissue processing a. Slides were incubated sequentially into Xylene; 15min – Xylene, 15min - Anhydrous ethanol, 5min - Anhydrous ethanol, 5min - 85% alcohol, 5min - 75% alcohol & 5min – wash in distilled water. b. 36. Antigen retrieval a. Tissue slides were incubated with citric acid (PH6.0) antigen retrieval buffer and microwaved for antigen retrieval (heated until boiled and then stopped heating) for 8min. Slides were then heated with medium power for 7min. During this process slides were kept from drying out. After cooling down at room temperature, slides were washed with PBS on shaker for 5min, repeated for 3 times. b. 37. Inhibition of endogenous peroxidase a. Slides were placed in 3% Hydrogen peroxide solution, and incubated for 10 min at room temperature without light exposure. Slides were then washed 3 times with PBS on a shaker for 5mins. b. 38. BSA Blocking a. Shortly after slides were dried, a PAP pen was used to draw circles around the tissue sections (and to prevent draining of the antibody solution). Inside the circles, BSA was used to cover the tissue evenly, blocking for 30min. b. 39. Primary antibody incubation After blocking solution was removed a 1:200 solution of primary antibody/PBS was added on the slide, and incubated overnight at 4°C. St John's Laboratory Ltd. www.stjohnslabs.com
Figure: Immunohistochemical analysis of paraffin embedded Mouse colon tissue. 1: GSK3β (phospho Ser9) Polyclonal Antibody was diluted at 1:200 (4 degree Celsius,overnight). 2: Sodium citrate pH 6.0 was used for antibody retrieval (>98 degree Celsius,20min). 3: Secondary antibody was diluted at 1:200 (room temperature, 30min). Negative control was used by secondary antibody only. Report Number 90284-u Host Rabbit Application IHC-P Clonality Polyclonal Model Number STJ90284 Clone ID NA Antibody Name Anti-Phospho-GSK3β (S9) antibody Testing Species MOUSE Testing Tissue COLON ANTIBODY VALIDATION REPORT a. (A small amount of distilled water was added into the incubation box to prevent evaporation of antibody). 29. Secondary antibody incubation a. Slides were washed 3 times, with PBS on a shaker for 5min. Shortly after the slides were dried the corresponding secondary antibody solution was added (HRP labelled), covering the tissues, and incubated at room temperature for 30min. b. 30. DAB staining a. Slides were washed 3 times, with PBS on a shaker for 5min. b. Shortly after, the slides were dried and fresh DAB staining buffer was added inside the circles. The staining time was adjusted under a microscope. Yellow-brown colour represented a positive result. Slides were washed with water to stop the staining. c. 31. Haematoxylin staining a. Haematoxylin was used to counter-staining for 1min, and then the slides were washed with water. 1% Hydrochloric acid and alcohol was added for several seconds and then washed with water. Ammonia was used to reveal blue colour, and then flushed with water. b. 32. Desolation and Clearing i. Slides were incubated sequentially into: 75% alcohol 5min, 85% alcohol 5min, Anhydrous ethanol - 5min, Anhydrous ethanol - 5min & Xylene - 5min. Shortly after slides were dried and neutral gum was used to seal the slides. ii. 33. Visualization a. Results were validated with microscope, and the slides were scanned. Paraffin-Embedded Immunohistochemistry Protocol 23. 24. Tissue processing a. Slides were incubated sequentially into Xylene; 15min – Xylene, 15min - Anhydrous ethanol, 5min - Anhydrous ethanol, 5min - 85% alcohol, 5min - 75% alcohol & 5min – wash in distilled water. b. 25. Antigen retrieval a. Tissue slides were incubated with citric acid (PH6.0) antigen retrieval buffer and microwaved for antigen retrieval (heated until boiled and then stopped heating) for 8min. Slides were then heated with medium power for 7min. During this process slides were kept from drying out. After cooling down at room temperature, slides were washed with PBS on shaker for 5min, repeated for 3 times. b. 26. Inhibition of endogenous peroxidase a. Slides were placed in 3% Hydrogen peroxide solution, and incubated for 10 min at room temperature without light exposure. Slides were then washed 3 times with PBS on a shaker for 5mins. b. 27. BSA Blocking a. Shortly after slides were dried, a PAP pen was used to draw circles around the tissue sections (and to prevent draining of the antibody solution). Inside the circles, BSA was used to cover the tissue evenly, blocking for 30min. b. 28. Primary antibody incubation After blocking solution was removed a 1:200 solution of primary antibody/PBS was added on the slide, and incubated overnight at 4°C. St John's Laboratory Ltd. www.stjohnslabs.com
Figure: Immunohistochemical analysis of paraffin embedded Mouse lung tissue. 1: GSK3β (phospho Ser9) Polyclonal Antibody was diluted at 1:200 (4 degree Celsius,overnight). 2: Sodium citrate pH 6.0 was used for antibody retrieval (>98 degree Celsius,20min). 3: Secondary antibody was diluted at 1:200 (room temperature, 30min). Negative control was used by secondary antibody only. Report Number 90284-v Host Rabbit Application IHC-P Clonality Polyclonal Model Number STJ90284 Clone ID NA Antibody Name Anti-Phospho-GSK3β (S9) antibody Testing Species MOUSE Testing Tissue LUNG ANTIBODY VALIDATION REPORT b. (A small amount of distilled water was added into the incubation box to prevent evaporation of antibody). 18. Secondary antibody incubation a. Slides were washed 3 times, with PBS on a shaker for 5min. Shortly after the slides were dried the corresponding secondary antibody solution was added (HRP labelled), covering the tissues, and incubated at room temperature for 30min. b. 19. DAB staining a. Slides were washed 3 times, with PBS on a shaker for 5min. b. Shortly after, the slides were dried and fresh DAB staining buffer was added inside the circles. The staining time was adjusted under a microscope. Yellow-brown colour represented a positive result. Slides were washed with water to stop the staining. c. 20. Haematoxylin staining a. Haematoxylin was used to counter-staining for 1min, and then the slides were washed with water. 1% Hydrochloric acid and alcohol was added for several seconds and then washed with water. Ammonia was used to reveal blue colour, and then flushed with water. b. 21. Desolation and Clearing i. Slides were incubated sequentially into: 75% alcohol 5min, 85% alcohol 5min, Anhydrous ethanol - 5min, Anhydrous ethanol - 5min & Xylene - 5min. Shortly after slides were dried and neutral gum was used to seal the slides. ii. 22. Visualization a. Results were validated with microscope, and the slides were scanned. Paraffin-Embedded Immunohistochemistry Protocol 12. 13. Tissue processing a. Slides were incubated sequentially into Xylene; 15min – Xylene, 15min - Anhydrous ethanol, 5min - Anhydrous ethanol, 5min - 85% alcohol, 5min - 75% alcohol & 5min – wash in distilled water. b. 14. Antigen retrieval a. Tissue slides were incubated with citric acid (PH6.0) antigen retrieval buffer and microwaved for antigen retrieval (heated until boiled and then stopped heating) for 8min. Slides were then heated with medium power for 7min. During this process slides were kept from drying out. After cooling down at room temperature, slides were washed with PBS on shaker for 5min, repeated for 3 times. b. 15. Inhibition of endogenous peroxidase a. Slides were placed in 3% Hydrogen peroxide solution, and incubated for 10 min at room temperature without light exposure. Slides were then washed 3 times with PBS on a shaker for 5mins. b. 16. BSA Blocking a. Shortly after slides were dried, a PAP pen was used to draw circles around the tissue sections (and to prevent draining of the antibody solution). Inside the circles, BSA was used to cover the tissue evenly, blocking for 30min. b. 17. Primary antibody incubation After blocking solution was removed a 1:200 solution of primary antibody/PBS was added on the slide, and incubated overnight at 4°C. St John's Laboratory Ltd. www.stjohnslabs.com
Figure: Immunohistochemical analysis of paraffin embedded Mouse kidney tissue. 1: GSK3β (phospho Ser9) Polyclonal Antibody was diluted at 1:200 (4 degree Celsius,overnight). 2: Sodium citrate pH 6.0 was used for antibody retrieval (>98 degree Celsius,20min). 3: Secondary antibody was diluted at 1:200 (room temperature, 30min). Negative control was used by secondary antibody only. Report Number 90284-w Host Rabbit Application IHC-P Clonality Polyclonal Model Number STJ90284 Clone ID NA Antibody Name Anti-Phospho-GSK3β (S9) antibody Testing Species MOUSE Testing Tissue KIDNEY ANTIBODY VALIDATION REPORT b. (A small amount of distilled water was added into the incubation box to prevent evaporation of antibody). 7. Secondary antibody incubation a. Slides were washed 3 times, with PBS on a shaker for 5min. Shortly after the slides were dried the corresponding secondary antibody solution was added (HRP labelled), covering the tissues, and incubated at room temperature for 30min. b. 8. DAB staining a. Slides were washed 3 times, with PBS on a shaker for 5min. b. Shortly after, the slides were dried and fresh DAB staining buffer was added inside the circles. The staining time was adjusted under a microscope. Yellow-brown colour represented a positive result. Slides were washed with water to stop the staining. c. 9. Haematoxylin staining a. Haematoxylin was used to counter-staining for 1min, and then the slides were washed with water. 1% Hydrochloric acid and alcohol was added for several seconds and then washed with water. Ammonia was used to reveal blue colour, and then flushed with water. b. 10. Desolation and Clearing i. Slides were incubated sequentially into: 75% alcohol 5min, 85% alcohol 5min, Anhydrous ethanol - 5min, Anhydrous ethanol - 5min & Xylene - 5min. Shortly after slides were dried and neutral gum was used to seal the slides. ii. 11. Visualization a. Results were validated with microscope, and the slides were scanned. Paraffin-Embedded Immunohistochemistry Protocol 1. 2. Tissue processing a. Slides were incubated sequentially into Xylene; 15min – Xylene, 15min - Anhydrous ethanol, 5min - Anhydrous ethanol, 5min - 85% alcohol, 5min - 75% alcohol & 5min – wash in distilled water. b. 3. Antigen retrieval a. Tissue slides were incubated with citric acid (PH6.0) antigen retrieval buffer and microwaved for antigen retrieval (heated until boiled and then stopped heating) for 8min. Slides were then heated with medium power for 7min. During this process slides were kept from drying out. After cooling down at room temperature, slides were washed with PBS on shaker for 5min, repeated for 3 times. b. 4. Inhibition of endogenous peroxidase a. Slides were placed in 3% Hydrogen peroxide solution, and incubated for 10 min at room temperature without light exposure. Slides were then washed 3 times with PBS on a shaker for 5mins. b. 5. BSA Blocking a. Shortly after slides were dried, a PAP pen was used to draw circles around the tissue sections (and to prevent draining of the antibody solution). Inside the circles, BSA was used to cover the tissue evenly, blocking for 30min. b. 6. Primary antibody incubation After blocking solution was removed a 1:200 solution of primary antibody/PBS was added on the slide, and incubated overnight at 4°C. St John's Laboratory Ltd. www.stjohnslabs.com
Figure: Immunohistochemical analysis of paraffin embedded Mouse brain tissue. 1: GSK3β (phospho Ser9) Polyclonal Antibody was diluted at 1:200 (4 degree Celsius,overnight). 2: Sodium citrate pH 6.0 was used for antibody retrieval (>98 degree Celsius,20min). 3: Secondary antibody was diluted at 1:200 (room temperature, 30min). Negative control was used by secondary antibody only. Report Number 90284-x Host Rabbit Application IHC-P Clonality Polyclonal Model Number STJ90284 Clone ID NA Antibody Name Anti-Phospho-GSK3β (S9) antibody Testing Species MOUSE Testing Tissue BRAIN ANTIBODY VALIDATION REPORT c. (A small amount of distilled water was added into the incubation box to prevent evaporation of antibody). 254. Secondary antibody incubation a. Slides were washed 3 times, with PBS on a shaker for 5min. Shortly after the slides were dried the corresponding secondary antibody solution was added (HRP labelled), covering the tissues, and incubated at room temperature for 30min. b. 255. DAB staining a. Slides were washed 3 times, with PBS on a shaker for 5min. b. Shortly after, the slides were dried and fresh DAB staining buffer was added inside the circles. The staining time was adjusted under a microscope. Yellow-brown colour represented a positive result. Slides were washed with water to stop the staining. c. 256. Haematoxylin staining a. Haematoxylin was used to counter-staining for 1min, and then the slides were washed with water. 1% Hydrochloric acid and alcohol was added for several seconds and then washed with water. Ammonia was used to reveal blue colour, and then flushed with water. b. 257. Desolation and Clearing i. Slides were incubated sequentially into: 75% alcohol 5min, 85% alcohol 5min, Anhydrous ethanol - 5min, Anhydrous ethanol - 5min & Xylene - 5min. Shortly after slides were dried and neutral gum was used to seal the slides. ii. 258. Visualization a. Results were validated with microscope, and the slides were scanned. Paraffin-Embedded Immunohistochemistry Protocol 259. 260. Tissue processing a. Slides were incubated sequentially into Xylene; 15min – Xylene, 15min - Anhydrous ethanol, 5min - Anhydrous ethanol, 5min - 85% alcohol, 5min - 75% alcohol & 5min – wash in distilled water. b. 261. Antigen retrieval a. Tissue slides were incubated with citric acid (PH6.0) antigen retrieval buffer and microwaved for antigen retrieval (heated until boiled and then stopped heating) for 8min. Slides were then heated with medium power for 7min. During this process slides were kept from drying out. After cooling down at room temperature, slides were washed with PBS on shaker for 5min, repeated for 3 times. b. 262. Inhibition of endogenous peroxidase a. Slides were placed in 3% Hydrogen peroxide solution, and incubated for 10 min at room temperature without light exposure. Slides were then washed 3 times with PBS on a shaker for 5mins. b. 263. BSA Blocking a. Shortly after slides were dried, a PAP pen was used to draw circles around the tissue sections (and to prevent draining of the antibody solution). Inside the circles, BSA was used to cover the tissue evenly, blocking for 30min. b. 264. Primary antibody incubation After blocking solution was removed a 1:200 solution of primary antibody/PBS was added on the slide, and incubated overnight at 4°C. St John's Laboratory Ltd. www.stjohnslabs.com
Figure: Immunohistochemical analysis of paraffin embedded Mouse spleen tissue. 1: GSK3β (phospho Ser9) Polyclonal Antibody was diluted at 1:200 (4 degree Celsius,overnight). 2: Sodium citrate pH 6.0 was used for antibody retrieval (>98 degree Celsius,20min). 3: Secondary antibody was diluted at 1:200 (room temperature, 30min). Negative control was used by secondary antibody only. Report Number 90284-y Host Rabbit Application IHC-P Clonality Polyclonal Model Number STJ90284 Clone ID NA Antibody Name Anti-Phospho-GSK3β (S9) antibody Testing Species MOUSE Testing Tissue SPLEEN ANTIBODY VALIDATION REPORT b. (A small amount of distilled water was added into the incubation box to prevent evaporation of antibody). 265. Secondary antibody incubation a. Slides were washed 3 times, with PBS on a shaker for 5min. Shortly after the slides were dried the corresponding secondary antibody solution was added (HRP labelled), covering the tissues, and incubated at room temperature for 30min. b. 266. DAB staining a. Slides were washed 3 times, with PBS on a shaker for 5min. b. Shortly after, the slides were dried and fresh DAB staining buffer was added inside the circles. The staining time was adjusted under a microscope. Yellow-brown colour represented a positive result. Slides were washed with water to stop the staining. c. 267. Haematoxylin staining a. Haematoxylin was used to counter-staining for 1min, and then the slides were washed with water. 1% Hydrochloric acid and alcohol was added for several seconds and then washed with water. Ammonia was used to reveal blue colour, and then flushed with water. b. 268. Desolation and Clearing i. Slides were incubated sequentially into: 75% alcohol 5min, 85% alcohol 5min, Anhydrous ethanol - 5min, Anhydrous ethanol - 5min & Xylene - 5min. Shortly after slides were dried and neutral gum was used to seal the slides. ii. 269. Visualization a. Results were validated with microscope, and the slides were scanned. Paraffin-Embedded Immunohistochemistry Protocol 270. 271. Tissue processing a. Slides were incubated sequentially into Xylene; 15min – Xylene, 15min - Anhydrous ethanol, 5min - Anhydrous ethanol, 5min - 85% alcohol, 5min - 75% alcohol & 5min – wash in distilled water. b. 272. Antigen retrieval a. Tissue slides were incubated with citric acid (PH6.0) antigen retrieval buffer and microwaved for antigen retrieval (heated until boiled and then stopped heating) for 8min. Slides were then heated with medium power for 7min. During this process slides were kept from drying out. After cooling down at room temperature, slides were washed with PBS on shaker for 5min, repeated for 3 times. b. 273. Inhibition of endogenous peroxidase a. Slides were placed in 3% Hydrogen peroxide solution, and incubated for 10 min at room temperature without light exposure. Slides were then washed 3 times with PBS on a shaker for 5mins. b. 274. BSA Blocking a. Shortly after slides were dried, a PAP pen was used to draw circles around the tissue sections (and to prevent draining of the antibody solution). Inside the circles, BSA was used to cover the tissue evenly, blocking for 30min. b. 275. Primary antibody incubation After blocking solution was removed a 1:200 solution of primary antibody/PBS was added on the slide, and incubated overnight at 4°C. St John's Laboratory Ltd. www.stjohnslabs.com

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  • 1. Figure: Immunohistochemical analysis of paraffin embedded Human uterus tissue. 1: GSK3β (phospho Ser9) Polyclonal Antibody was diluted at 1:200 (4 degree Celsius,overnight). 2: Sodium citrate pH 6.0 was used for antibody retrieval (>98 degree Celsius,20min). 3: Secondary antibody was diluted at 1:200 (room temperature, 30min). Negative control was used by secondary antibody only. Report Number 90284-a Host Rabbit Application IHC-P Clonality Polyclonal Model Number STJ90284 Clone ID NA Antibody Name Anti-Phospho-GSK3β (S9) antibody Testing Species HUMAN Testing Tissue UTERUS ANTIBODY VALIDATION REPORT a. (A small amount of distilled water was added into the incubation box to prevent evaporation of antibody). 249. Secondary antibody incubation a. Slides were washed 3 times, with PBS on a shaker for 5min. Shortly after the slides were dried the corresponding secondary antibody solution was added (HRP labelled), covering the tissues, and incubated at room temperature for 30min. b. 250. DAB staining a. Slides were washed 3 times, with PBS on a shaker for 5min. b. Shortly after, the slides were dried and fresh DAB staining buffer was added inside the circles. The staining time was adjusted under a microscope. Yellow-brown colour represented a positive result. Slides were washed with water to stop the staining. c. 251. Haematoxylin staining a. Haematoxylin was used to counter-staining for 1min, and then the slides were washed with water. 1% Hydrochloric acid and alcohol was added for several seconds and then washed with water. Ammonia was used to reveal blue colour, and then flushed with water. b. 252. Desolation and Clearing i. Slides were incubated sequentially into: 75% alcohol 5min, 85% alcohol 5min, Anhydrous ethanol - 5min, Anhydrous ethanol - 5min & Xylene - 5min. Shortly after slides were dried and neutral gum was used to seal the slides. ii. 253. Visualization a. Results were validated with microscope, and the slides were scanned. Paraffin-Embedded Immunohistochemistry Protocol 243. 244. Tissue processing a. Slides were incubated sequentially into Xylene; 15min – Xylene, 15min - Anhydrous ethanol, 5min - Anhydrous ethanol, 5min - 85% alcohol, 5min - 75% alcohol & 5min – wash in distilled water. b. 245. Antigen retrieval a. Tissue slides were incubated with citric acid (PH6.0) antigen retrieval buffer and microwaved for antigen retrieval (heated until boiled and then stopped heating) for 8min. Slides were then heated with medium power for 7min. During this process slides were kept from drying out. After cooling down at room temperature, slides were washed with PBS on shaker for 5min, repeated for 3 times. b. 246. Inhibition of endogenous peroxidase a. Slides were placed in 3% Hydrogen peroxide solution, and incubated for 10 min at room temperature without light exposure. Slides were then washed 3 times with PBS on a shaker for 5mins. b. 247. BSA Blocking a. Shortly after slides were dried, a PAP pen was used to draw circles around the tissue sections (and to prevent draining of the antibody solution). Inside the circles, BSA was used to cover the tissue evenly, blocking for 30min. b. 248. Primary antibody incubation After blocking solution was removed a 1:200 solution of primary antibody/PBS was added on the slide, and incubated overnight at 4°C. St John's Laboratory Ltd. www.stjohnslabs.com
  • 2. Figure: Immunohistochemical analysis of paraffin embedded Human uterus cancer tissue. 1: GSK3β (phospho Ser9) Polyclonal Antibody was diluted at 1:200 (4 degree Celsius,overnight). 2: Sodium citrate pH 6.0 was used for antibody retrieval (>98 degree Celsius,20min). 3: Secondary antibody was diluted at 1:200 (room temperature, 30min). Negative control was used by secondary antibody only. Report Number 90284-b Host Rabbit Application IHC-P Clonality Polyclonal Model Number STJ90284 Clone ID NA Antibody Name Anti-Phospho-GSK3β (S9) antibody Testing Species HUMAN Testing Tissue UTERUS CANCER ANTIBODY VALIDATION REPORT a. (A small amount of distilled water was added into the incubation box to prevent evaporation of antibody). 238. Secondary antibody incubation a. Slides were washed 3 times, with PBS on a shaker for 5min. Shortly after the slides were dried the corresponding secondary antibody solution was added (HRP labelled), covering the tissues, and incubated at room temperature for 30min. b. 239. DAB staining a. Slides were washed 3 times, with PBS on a shaker for 5min. b. Shortly after, the slides were dried and fresh DAB staining buffer was added inside the circles. The staining time was adjusted under a microscope. Yellow-brown colour represented a positive result. Slides were washed with water to stop the staining. c. 240. Haematoxylin staining a. Haematoxylin was used to counter-staining for 1min, and then the slides were washed with water. 1% Hydrochloric acid and alcohol was added for several seconds and then washed with water. Ammonia was used to reveal blue colour, and then flushed with water. b. 241. Desolation and Clearing i. Slides were incubated sequentially into: 75% alcohol 5min, 85% alcohol 5min, Anhydrous ethanol - 5min, Anhydrous ethanol - 5min & Xylene - 5min. Shortly after slides were dried and neutral gum was used to seal the slides. ii. 242. Visualization a. Results were validated with microscope, and the slides were scanned. Paraffin-Embedded Immunohistochemistry Protocol 232. 233. Tissue processing a. Slides were incubated sequentially into Xylene; 15min – Xylene, 15min - Anhydrous ethanol, 5min - Anhydrous ethanol, 5min - 85% alcohol, 5min - 75% alcohol & 5min – wash in distilled water. b. 234. Antigen retrieval a. Tissue slides were incubated with citric acid (PH6.0) antigen retrieval buffer and microwaved for antigen retrieval (heated until boiled and then stopped heating) for 8min. Slides were then heated with medium power for 7min. During this process slides were kept from drying out. After cooling down at room temperature, slides were washed with PBS on shaker for 5min, repeated for 3 times. b. 235. Inhibition of endogenous peroxidase a. Slides were placed in 3% Hydrogen peroxide solution, and incubated for 10 min at room temperature without light exposure. Slides were then washed 3 times with PBS on a shaker for 5mins. b. 236. BSA Blocking a. Shortly after slides were dried, a PAP pen was used to draw circles around the tissue sections (and to prevent draining of the antibody solution). Inside the circles, BSA was used to cover the tissue evenly, blocking for 30min. b. 237. Primary antibody incubation After blocking solution was removed a 1:200 solution of primary antibody/PBS was added on the slide, and incubated overnight at 4°C. St John's Laboratory Ltd. www.stjohnslabs.com
  • 3. Figure: Immunohistochemical analysis of paraffin embedded Human colon tissue. 1: GSK3β (phospho Ser9) Polyclonal Antibody was diluted at 1:200 (4 degree Celsius,overnight). 2: Sodium citrate pH 6.0 was used for antibody retrieval (>98 degree Celsius,20min). 3: Secondary antibody was diluted at 1:200 (room temperature, 30min). Negative control was used by secondary antibody only. Report Number 90284-c Host Rabbit Application IHC-P Clonality Polyclonal Model Number STJ90284 Clone ID NA Antibody Name Anti-Phospho-GSK3β (S9) antibody Testing Species HUMAN Testing Tissue COLON ANTIBODY VALIDATION REPORT a. (A small amount of distilled water was added into the incubation box to prevent evaporation of antibody). 227. Secondary antibody incubation a. Slides were washed 3 times, with PBS on a shaker for 5min. Shortly after the slides were dried the corresponding secondary antibody solution was added (HRP labelled), covering the tissues, and incubated at room temperature for 30min. b. 228. DAB staining a. Slides were washed 3 times, with PBS on a shaker for 5min. b. Shortly after, the slides were dried and fresh DAB staining buffer was added inside the circles. The staining time was adjusted under a microscope. Yellow-brown colour represented a positive result. Slides were washed with water to stop the staining. c. 229. Haematoxylin staining a. Haematoxylin was used to counter-staining for 1min, and then the slides were washed with water. 1% Hydrochloric acid and alcohol was added for several seconds and then washed with water. Ammonia was used to reveal blue colour, and then flushed with water. b. 230. Desolation and Clearing i. Slides were incubated sequentially into: 75% alcohol 5min, 85% alcohol 5min, Anhydrous ethanol - 5min, Anhydrous ethanol - 5min & Xylene - 5min. Shortly after slides were dried and neutral gum was used to seal the slides. ii. 231. Visualization a. Results were validated with microscope, and the slides were scanned. Paraffin-Embedded Immunohistochemistry Protocol 221. 222. Tissue processing a. Slides were incubated sequentially into Xylene; 15min – Xylene, 15min - Anhydrous ethanol, 5min - Anhydrous ethanol, 5min - 85% alcohol, 5min - 75% alcohol & 5min – wash in distilled water. b. 223. Antigen retrieval a. Tissue slides were incubated with citric acid (PH6.0) antigen retrieval buffer and microwaved for antigen retrieval (heated until boiled and then stopped heating) for 8min. Slides were then heated with medium power for 7min. During this process slides were kept from drying out. After cooling down at room temperature, slides were washed with PBS on shaker for 5min, repeated for 3 times. b. 224. Inhibition of endogenous peroxidase a. Slides were placed in 3% Hydrogen peroxide solution, and incubated for 10 min at room temperature without light exposure. Slides were then washed 3 times with PBS on a shaker for 5mins. b. 225. BSA Blocking a. Shortly after slides were dried, a PAP pen was used to draw circles around the tissue sections (and to prevent draining of the antibody solution). Inside the circles, BSA was used to cover the tissue evenly, blocking for 30min. b. 226. Primary antibody incubation After blocking solution was removed a 1:200 solution of primary antibody/PBS was added on the slide, and incubated overnight at 4°C. St John's Laboratory Ltd. www.stjohnslabs.com
  • 4. Figure: Immunohistochemical analysis of paraffin embedded Human liver tissue. 1: GSK3β (phospho Ser9) Polyclonal Antibody was diluted at 1:200 (4 degree Celsius,overnight). 2: Sodium citrate pH 6.0 was used for antibody retrieval (>98 degree Celsius,20min). 3: Secondary antibody was diluted at 1:200 (room temperature, 30min). Negative control was used by secondary antibody only. Report Number 90284-d Host Rabbit Application IHC-P Clonality Polyclonal Model Number STJ90284 Clone ID NA Antibody Name Anti-Phospho-GSK3β (S9) antibody Testing Species HUMAN Testing Tissue LIVER ANTIBODY VALIDATION REPORT a. (A small amount of distilled water was added into the incubation box to prevent evaporation of antibody). 216. Secondary antibody incubation a. Slides were washed 3 times, with PBS on a shaker for 5min. Shortly after the slides were dried the corresponding secondary antibody solution was added (HRP labelled), covering the tissues, and incubated at room temperature for 30min. b. 217. DAB staining a. Slides were washed 3 times, with PBS on a shaker for 5min. b. Shortly after, the slides were dried and fresh DAB staining buffer was added inside the circles. The staining time was adjusted under a microscope. Yellow-brown colour represented a positive result. Slides were washed with water to stop the staining. c. 218. Haematoxylin staining a. Haematoxylin was used to counter-staining for 1min, and then the slides were washed with water. 1% Hydrochloric acid and alcohol was added for several seconds and then washed with water. Ammonia was used to reveal blue colour, and then flushed with water. b. 219. Desolation and Clearing i. Slides were incubated sequentially into: 75% alcohol 5min, 85% alcohol 5min, Anhydrous ethanol - 5min, Anhydrous ethanol - 5min & Xylene - 5min. Shortly after slides were dried and neutral gum was used to seal the slides. ii. 220. Visualization a. Results were validated with microscope, and the slides were scanned. Paraffin-Embedded Immunohistochemistry Protocol 210. 211. Tissue processing a. Slides were incubated sequentially into Xylene; 15min – Xylene, 15min - Anhydrous ethanol, 5min - Anhydrous ethanol, 5min - 85% alcohol, 5min - 75% alcohol & 5min – wash in distilled water. b. 212. Antigen retrieval a. Tissue slides were incubated with citric acid (PH6.0) antigen retrieval buffer and microwaved for antigen retrieval (heated until boiled and then stopped heating) for 8min. Slides were then heated with medium power for 7min. During this process slides were kept from drying out. After cooling down at room temperature, slides were washed with PBS on shaker for 5min, repeated for 3 times. b. 213. Inhibition of endogenous peroxidase a. Slides were placed in 3% Hydrogen peroxide solution, and incubated for 10 min at room temperature without light exposure. Slides were then washed 3 times with PBS on a shaker for 5mins. b. 214. BSA Blocking a. Shortly after slides were dried, a PAP pen was used to draw circles around the tissue sections (and to prevent draining of the antibody solution). Inside the circles, BSA was used to cover the tissue evenly, blocking for 30min. b. 215. Primary antibody incubation After blocking solution was removed a 1:200 solution of primary antibody/PBS was added on the slide, and incubated overnight at 4°C. St John's Laboratory Ltd. www.stjohnslabs.com
  • 5. Figure: Immunohistochemical analysis of paraffin embedded Human liver cancer tissue. 1: GSK3β (phospho Ser9) Polyclonal Antibody was diluted at 1:200 (4 degree Celsius,overnight). 2: Sodium citrate pH 6.0 was used for antibody retrieval (>98 degree Celsius,20min). 3: Secondary antibody was diluted at 1:200 (room temperature, 30min). Negative control was used by secondary antibody only. Report Number 90284-e Host Rabbit Application IHC-P Clonality Polyclonal Model Number STJ90284 Clone ID NA Antibody Name Anti-Phospho-GSK3β (S9) antibody Testing Species HUMAN Testing Tissue LIVER CANCER ANTIBODY VALIDATION REPORT a. (A small amount of distilled water was added into the incubation box to prevent evaporation of antibody). 205. Secondary antibody incubation a. Slides were washed 3 times, with PBS on a shaker for 5min. Shortly after the slides were dried the corresponding secondary antibody solution was added (HRP labelled), covering the tissues, and incubated at room temperature for 30min. b. 206. DAB staining a. Slides were washed 3 times, with PBS on a shaker for 5min. b. Shortly after, the slides were dried and fresh DAB staining buffer was added inside the circles. The staining time was adjusted under a microscope. Yellow-brown colour represented a positive result. Slides were washed with water to stop the staining. c. 207. Haematoxylin staining a. Haematoxylin was used to counter-staining for 1min, and then the slides were washed with water. 1% Hydrochloric acid and alcohol was added for several seconds and then washed with water. Ammonia was used to reveal blue colour, and then flushed with water. b. 208. Desolation and Clearing i. Slides were incubated sequentially into: 75% alcohol 5min, 85% alcohol 5min, Anhydrous ethanol - 5min, Anhydrous ethanol - 5min & Xylene - 5min. Shortly after slides were dried and neutral gum was used to seal the slides. ii. 209. Visualization a. Results were validated with microscope, and the slides were scanned. Paraffin-Embedded Immunohistochemistry Protocol 199. 200. Tissue processing a. Slides were incubated sequentially into Xylene; 15min – Xylene, 15min - Anhydrous ethanol, 5min - Anhydrous ethanol, 5min - 85% alcohol, 5min - 75% alcohol & 5min – wash in distilled water. b. 201. Antigen retrieval a. Tissue slides were incubated with citric acid (PH6.0) antigen retrieval buffer and microwaved for antigen retrieval (heated until boiled and then stopped heating) for 8min. Slides were then heated with medium power for 7min. During this process slides were kept from drying out. After cooling down at room temperature, slides were washed with PBS on shaker for 5min, repeated for 3 times. b. 202. Inhibition of endogenous peroxidase a. Slides were placed in 3% Hydrogen peroxide solution, and incubated for 10 min at room temperature without light exposure. Slides were then washed 3 times with PBS on a shaker for 5mins. b. 203. BSA Blocking a. Shortly after slides were dried, a PAP pen was used to draw circles around the tissue sections (and to prevent draining of the antibody solution). Inside the circles, BSA was used to cover the tissue evenly, blocking for 30min. b. 204. Primary antibody incubation After blocking solution was removed a 1:200 solution of primary antibody/PBS was added on the slide, and incubated overnight at 4°C. St John's Laboratory Ltd. www.stjohnslabs.com
  • 6. Figure: Immunohistochemical analysis of paraffin embedded Human lung tissue. 1: GSK3β (phospho Ser9) Polyclonal Antibody was diluted at 1:200 (4 degree Celsius,overnight). 2: Sodium citrate pH 6.0 was used for antibody retrieval (>98 degree Celsius,20min). 3: Secondary antibody was diluted at 1:200 (room temperature, 30min). Negative control was used by secondary antibody only. Report Number 90284-f Host Rabbit Application IHC-P Clonality Polyclonal Model Number STJ90284 Clone ID NA Antibody Name Anti-Phospho-GSK3β (S9) antibody Testing Species HUMAN Testing Tissue LUNG ANTIBODY VALIDATION REPORT a. (A small amount of distilled water was added into the incubation box to prevent evaporation of antibody). 194. Secondary antibody incubation a. Slides were washed 3 times, with PBS on a shaker for 5min. Shortly after the slides were dried the corresponding secondary antibody solution was added (HRP labelled), covering the tissues, and incubated at room temperature for 30min. b. 195. DAB staining a. Slides were washed 3 times, with PBS on a shaker for 5min. b. Shortly after, the slides were dried and fresh DAB staining buffer was added inside the circles. The staining time was adjusted under a microscope. Yellow-brown colour represented a positive result. Slides were washed with water to stop the staining. c. 196. Haematoxylin staining a. Haematoxylin was used to counter-staining for 1min, and then the slides were washed with water. 1% Hydrochloric acid and alcohol was added for several seconds and then washed with water. Ammonia was used to reveal blue colour, and then flushed with water. b. 197. Desolation and Clearing i. Slides were incubated sequentially into: 75% alcohol 5min, 85% alcohol 5min, Anhydrous ethanol - 5min, Anhydrous ethanol - 5min & Xylene - 5min. Shortly after slides were dried and neutral gum was used to seal the slides. ii. 198. Visualization a. Results were validated with microscope, and the slides were scanned. Paraffin-Embedded Immunohistochemistry Protocol 188. 189. Tissue processing a. Slides were incubated sequentially into Xylene; 15min – Xylene, 15min - Anhydrous ethanol, 5min - Anhydrous ethanol, 5min - 85% alcohol, 5min - 75% alcohol & 5min – wash in distilled water. b. 190. Antigen retrieval a. Tissue slides were incubated with citric acid (PH6.0) antigen retrieval buffer and microwaved for antigen retrieval (heated until boiled and then stopped heating) for 8min. Slides were then heated with medium power for 7min. During this process slides were kept from drying out. After cooling down at room temperature, slides were washed with PBS on shaker for 5min, repeated for 3 times. b. 191. Inhibition of endogenous peroxidase a. Slides were placed in 3% Hydrogen peroxide solution, and incubated for 10 min at room temperature without light exposure. Slides were then washed 3 times with PBS on a shaker for 5mins. b. 192. BSA Blocking a. Shortly after slides were dried, a PAP pen was used to draw circles around the tissue sections (and to prevent draining of the antibody solution). Inside the circles, BSA was used to cover the tissue evenly, blocking for 30min. b. 193. Primary antibody incubation After blocking solution was removed a 1:200 solution of primary antibody/PBS was added on the slide, and incubated overnight at 4°C. St John's Laboratory Ltd. www.stjohnslabs.com
  • 7. Figure: Immunohistochemical analysis of paraffin embedded Human stomach tissue. 1: GSK3 β (phospho Ser9) Polyclonal Antibody was diluted at 1:200 (4 degree Celsius,overnight). 2: Sodium citrate pH 6.0 was used for antibody retrieval (>98 degree Celsius,20min). 3: Secondary antibody was diluted at 1:200 (room temperature, 30min). Negative control was used by secondary antibody only. Report Number 90284-g Host Rabbit Application IHC-P Clonality Polyclonal Model Number STJ90284 Clone ID NA Antibody Name Anti-Phospho-GSK3β (S9) antibody Testing Species HUMAN Testing Tissue STOMACH ANTIBODY VALIDATION REPORT a. (A small amount of distilled water was added into the incubation box to prevent evaporation of antibody). 183. Secondary antibody incubation a. Slides were washed 3 times, with PBS on a shaker for 5min. Shortly after the slides were dried the corresponding secondary antibody solution was added (HRP labelled), covering the tissues, and incubated at room temperature for 30min. b. 184. DAB staining a. Slides were washed 3 times, with PBS on a shaker for 5min. b. Shortly after, the slides were dried and fresh DAB staining buffer was added inside the circles. The staining time was adjusted under a microscope. Yellow-brown colour represented a positive result. Slides were washed with water to stop the staining. c. 185. Haematoxylin staining a. Haematoxylin was used to counter-staining for 1min, and then the slides were washed with water. 1% Hydrochloric acid and alcohol was added for several seconds and then washed with water. Ammonia was used to reveal blue colour, and then flushed with water. b. 186. Desolation and Clearing i. Slides were incubated sequentially into: 75% alcohol 5min, 85% alcohol 5min, Anhydrous ethanol - 5min, Anhydrous ethanol - 5min & Xylene - 5min. Shortly after slides were dried and neutral gum was used to seal the slides. ii. 187. Visualization a. Results were validated with microscope, and the slides were scanned. Paraffin-Embedded Immunohistochemistry Protocol 177. 178. Tissue processing a. Slides were incubated sequentially into Xylene; 15min – Xylene, 15min - Anhydrous ethanol, 5min - Anhydrous ethanol, 5min - 85% alcohol, 5min - 75% alcohol & 5min – wash in distilled water. b. 179. Antigen retrieval a. Tissue slides were incubated with citric acid (PH6.0) antigen retrieval buffer and microwaved for antigen retrieval (heated until boiled and then stopped heating) for 8min. Slides were then heated with medium power for 7min. During this process slides were kept from drying out. After cooling down at room temperature, slides were washed with PBS on shaker for 5min, repeated for 3 times. b. 180. Inhibition of endogenous peroxidase a. Slides were placed in 3% Hydrogen peroxide solution, and incubated for 10 min at room temperature without light exposure. Slides were then washed 3 times with PBS on a shaker for 5mins. b. 181. BSA Blocking a. Shortly after slides were dried, a PAP pen was used to draw circles around the tissue sections (and to prevent draining of the antibody solution). Inside the circles, BSA was used to cover the tissue evenly, blocking for 30min. b. 182. Primary antibody incubation After blocking solution was removed a 1:200 solution of primary antibody/PBS was added on the slide, and incubated overnight at 4°C. St John's Laboratory Ltd. www.stjohnslabs.com
  • 8. Figure: Immunohistochemical analysis of paraffin embedded Human stomach cancer tissue. 1: GSK3β (phospho Ser9) Polyclonal Antibody was diluted at 1:200 (4 degree Celsius,overnight). 2: Sodium citrate pH 6.0 was used for antibody retrieval (>98 degree Celsius,20min). 3: Secondary antibody was diluted at 1:200 (room temperature, 30min). Negative control was used by secondary antibody only. Report Number 90284-h Host Rabbit Application IHC-P Clonality Polyclonal Model Number STJ90284 Clone ID NA Antibody Name Anti-Phospho-GSK3β (S9) antibody Testing Species HUMAN Testing Tissue STOMACH CANCER ANTIBODY VALIDATION REPORT a. (A small amount of distilled water was added into the incubation box to prevent evaporation of antibody). 172. Secondary antibody incubation a. Slides were washed 3 times, with PBS on a shaker for 5min. Shortly after the slides were dried the corresponding secondary antibody solution was added (HRP labelled), covering the tissues, and incubated at room temperature for 30min. b. 173. DAB staining a. Slides were washed 3 times, with PBS on a shaker for 5min. b. Shortly after, the slides were dried and fresh DAB staining buffer was added inside the circles. The staining time was adjusted under a microscope. Yellow-brown colour represented a positive result. Slides were washed with water to stop the staining. c. 174. Haematoxylin staining a. Haematoxylin was used to counter-staining for 1min, and then the slides were washed with water. 1% Hydrochloric acid and alcohol was added for several seconds and then washed with water. Ammonia was used to reveal blue colour, and then flushed with water. b. 175. Desolation and Clearing i. Slides were incubated sequentially into: 75% alcohol 5min, 85% alcohol 5min, Anhydrous ethanol - 5min, Anhydrous ethanol - 5min & Xylene - 5min. Shortly after slides were dried and neutral gum was used to seal the slides. ii. 176. Visualization a. Results were validated with microscope, and the slides were scanned. Paraffin-Embedded Immunohistochemistry Protocol 166. 167. Tissue processing a. Slides were incubated sequentially into Xylene; 15min – Xylene, 15min - Anhydrous ethanol, 5min - Anhydrous ethanol, 5min - 85% alcohol, 5min - 75% alcohol & 5min – wash in distilled water. b. 168. Antigen retrieval a. Tissue slides were incubated with citric acid (PH6.0) antigen retrieval buffer and microwaved for antigen retrieval (heated until boiled and then stopped heating) for 8min. Slides were then heated with medium power for 7min. During this process slides were kept from drying out. After cooling down at room temperature, slides were washed with PBS on shaker for 5min, repeated for 3 times. b. 169. Inhibition of endogenous peroxidase a. Slides were placed in 3% Hydrogen peroxide solution, and incubated for 10 min at room temperature without light exposure. Slides were then washed 3 times with PBS on a shaker for 5mins. b. 170. BSA Blocking a. Shortly after slides were dried, a PAP pen was used to draw circles around the tissue sections (and to prevent draining of the antibody solution). Inside the circles, BSA was used to cover the tissue evenly, blocking for 30min. b. 171. Primary antibody incubation After blocking solution was removed a 1:200 solution of primary antibody/PBS was added on the slide, and incubated overnight at 4°C. St John's Laboratory Ltd. www.stjohnslabs.com
  • 9. Figure: Immunohistochemical analysis of paraffin embedded Human appendix tissue. 1: GSK3β (phospho Ser9) Polyclonal Antibody was diluted at 1:200 (4 degree Celsius,overnight). 2: Sodium citrate pH 6.0 was used for antibody retrieval (>98 degree Celsius,20min). 3: Secondary antibody was diluted at 1:200 (room temperature, 30min). Negative control was used by secondary antibody only. Report Number 90284-i Host Rabbit Application IHC-P Clonality Polyclonal Model Number STJ90284 Clone ID NA Antibody Name Anti-Phospho-GSK3β (S9) antibody Testing Species HUMAN Testing Tissue APPENDIX ANTIBODY VALIDATION REPORT a. (A small amount of distilled water was added into the incubation box to prevent evaporation of antibody). 161. Secondary antibody incubation a. Slides were washed 3 times, with PBS on a shaker for 5min. Shortly after the slides were dried the corresponding secondary antibody solution was added (HRP labelled), covering the tissues, and incubated at room temperature for 30min. b. 162. DAB staining a. Slides were washed 3 times, with PBS on a shaker for 5min. b. Shortly after, the slides were dried and fresh DAB staining buffer was added inside the circles. The staining time was adjusted under a microscope. Yellow-brown colour represented a positive result. Slides were washed with water to stop the staining. c. 163. Haematoxylin staining a. Haematoxylin was used to counter-staining for 1min, and then the slides were washed with water. 1% Hydrochloric acid and alcohol was added for several seconds and then washed with water. Ammonia was used to reveal blue colour, and then flushed with water. b. 164. Desolation and Clearing i. Slides were incubated sequentially into: 75% alcohol 5min, 85% alcohol 5min, Anhydrous ethanol - 5min, Anhydrous ethanol - 5min & Xylene - 5min. Shortly after slides were dried and neutral gum was used to seal the slides. ii. 165. Visualization a. Results were validated with microscope, and the slides were scanned. Paraffin-Embedded Immunohistochemistry Protocol 155. 156. Tissue processing a. Slides were incubated sequentially into Xylene; 15min – Xylene, 15min - Anhydrous ethanol, 5min - Anhydrous ethanol, 5min - 85% alcohol, 5min - 75% alcohol & 5min – wash in distilled water. b. 157. Antigen retrieval a. Tissue slides were incubated with citric acid (PH6.0) antigen retrieval buffer and microwaved for antigen retrieval (heated until boiled and then stopped heating) for 8min. Slides were then heated with medium power for 7min. During this process slides were kept from drying out. After cooling down at room temperature, slides were washed with PBS on shaker for 5min, repeated for 3 times. b. 158. Inhibition of endogenous peroxidase a. Slides were placed in 3% Hydrogen peroxide solution, and incubated for 10 min at room temperature without light exposure. Slides were then washed 3 times with PBS on a shaker for 5mins. b. 159. BSA Blocking a. Shortly after slides were dried, a PAP pen was used to draw circles around the tissue sections (and to prevent draining of the antibody solution). Inside the circles, BSA was used to cover the tissue evenly, blocking for 30min. b. 160. Primary antibody incubation After blocking solution was removed a 1:200 solution of primary antibody/PBS was added on the slide, and incubated overnight at 4°C. St John's Laboratory Ltd. www.stjohnslabs.com
  • 10. Figure: Immunohistochemical analysis of paraffin embedded Rat heart tissue. 1: GSK3β (phospho Ser9) Polyclonal Antibody was diluted at 1:200 (4 degree Celsius,overnight). 2: Sodium citrate pH 6.0 was used for antibody retrieval (>98 degree Celsius,20min). 3: Secondary antibody was diluted at 1:200 (room temperature, 30min). Negative control was used by secondary antibody only. Report Number 90284-j Host Rabbit Application IHC-P Clonality Polyclonal Model Number STJ90284 Clone ID NA Antibody Name Anti-Phospho-GSK3β (S9) antibody Testing Species RAT Testing Tissue HEART ANTIBODY VALIDATION REPORT a. (A small amount of distilled water was added into the incubation box to prevent evaporation of antibody). 150. Secondary antibody incubation a. Slides were washed 3 times, with PBS on a shaker for 5min. Shortly after the slides were dried the corresponding secondary antibody solution was added (HRP labelled), covering the tissues, and incubated at room temperature for 30min. b. 151. DAB staining a. Slides were washed 3 times, with PBS on a shaker for 5min. b. Shortly after, the slides were dried and fresh DAB staining buffer was added inside the circles. The staining time was adjusted under a microscope. Yellow-brown colour represented a positive result. Slides were washed with water to stop the staining. c. 152. Haematoxylin staining a. Haematoxylin was used to counter-staining for 1min, and then the slides were washed with water. 1% Hydrochloric acid and alcohol was added for several seconds and then washed with water. Ammonia was used to reveal blue colour, and then flushed with water. b. 153. Desolation and Clearing i. Slides were incubated sequentially into: 75% alcohol 5min, 85% alcohol 5min, Anhydrous ethanol - 5min, Anhydrous ethanol - 5min & Xylene - 5min. Shortly after slides were dried and neutral gum was used to seal the slides. ii. 154. Visualization a. Results were validated with microscope, and the slides were scanned. Paraffin-Embedded Immunohistochemistry Protocol 144. 145. Tissue processing a. Slides were incubated sequentially into Xylene; 15min – Xylene, 15min - Anhydrous ethanol, 5min - Anhydrous ethanol, 5min - 85% alcohol, 5min - 75% alcohol & 5min – wash in distilled water. b. 146. Antigen retrieval a. Tissue slides were incubated with citric acid (PH6.0) antigen retrieval buffer and microwaved for antigen retrieval (heated until boiled and then stopped heating) for 8min. Slides were then heated with medium power for 7min. During this process slides were kept from drying out. After cooling down at room temperature, slides were washed with PBS on shaker for 5min, repeated for 3 times. b. 147. Inhibition of endogenous peroxidase a. Slides were placed in 3% Hydrogen peroxide solution, and incubated for 10 min at room temperature without light exposure. Slides were then washed 3 times with PBS on a shaker for 5mins. b. 148. BSA Blocking a. Shortly after slides were dried, a PAP pen was used to draw circles around the tissue sections (and to prevent draining of the antibody solution). Inside the circles, BSA was used to cover the tissue evenly, blocking for 30min. b. 149. Primary antibody incubation After blocking solution was removed a 1:200 solution of primary antibody/PBS was added on the slide, and incubated overnight at 4°C. St John's Laboratory Ltd. www.stjohnslabs.com
  • 11. Figure: Immunohistochemical analysis of paraffin embedded Rat testis tissue. 1: GSK3β (phospho Ser9) Polyclonal Antibody was diluted at 1:200 (4 degree Celsius,overnight). 2: Sodium citrate pH 6.0 was used for antibody retrieval (>98 degree Celsius,20min). 3: Secondary antibody was diluted at 1:200 (room temperature, 30min). Negative control was used by secondary antibody only. Report Number 90284-k Host Rabbit Application IHC-P Clonality Polyclonal Model Number STJ90284 Clone ID NA Antibody Name Anti-Phospho-GSK3β (S9) antibody Testing Species RAT Testing Tissue TESTIS ANTIBODY VALIDATION REPORT a. (A small amount of distilled water was added into the incubation box to prevent evaporation of antibody). 139. Secondary antibody incubation a. Slides were washed 3 times, with PBS on a shaker for 5min. Shortly after the slides were dried the corresponding secondary antibody solution was added (HRP labelled), covering the tissues, and incubated at room temperature for 30min. b. 140. DAB staining a. Slides were washed 3 times, with PBS on a shaker for 5min. b. Shortly after, the slides were dried and fresh DAB staining buffer was added inside the circles. The staining time was adjusted under a microscope. Yellow-brown colour represented a positive result. Slides were washed with water to stop the staining. c. 141. Haematoxylin staining a. Haematoxylin was used to counter-staining for 1min, and then the slides were washed with water. 1% Hydrochloric acid and alcohol was added for several seconds and then washed with water. Ammonia was used to reveal blue colour, and then flushed with water. b. 142. Desolation and Clearing i. Slides were incubated sequentially into: 75% alcohol 5min, 85% alcohol 5min, Anhydrous ethanol - 5min, Anhydrous ethanol - 5min & Xylene - 5min. Shortly after slides were dried and neutral gum was used to seal the slides. ii. 143. Visualization a. Results were validated with microscope, and the slides were scanned. Paraffin-Embedded Immunohistochemistry Protocol 133. 134. Tissue processing a. Slides were incubated sequentially into Xylene; 15min – Xylene, 15min - Anhydrous ethanol, 5min - Anhydrous ethanol, 5min - 85% alcohol, 5min - 75% alcohol & 5min – wash in distilled water. b. 135. Antigen retrieval a. Tissue slides were incubated with citric acid (PH6.0) antigen retrieval buffer and microwaved for antigen retrieval (heated until boiled and then stopped heating) for 8min. Slides were then heated with medium power for 7min. During this process slides were kept from drying out. After cooling down at room temperature, slides were washed with PBS on shaker for 5min, repeated for 3 times. b. 136. Inhibition of endogenous peroxidase a. Slides were placed in 3% Hydrogen peroxide solution, and incubated for 10 min at room temperature without light exposure. Slides were then washed 3 times with PBS on a shaker for 5mins. b. 137. BSA Blocking a. Shortly after slides were dried, a PAP pen was used to draw circles around the tissue sections (and to prevent draining of the antibody solution). Inside the circles, BSA was used to cover the tissue evenly, blocking for 30min. b. 138. Primary antibody incubation After blocking solution was removed a 1:200 solution of primary antibody/PBS was added on the slide, and incubated overnight at 4°C. St John's Laboratory Ltd. www.stjohnslabs.com
  • 12. Figure: Immunohistochemical analysis of paraffin embedded Rat liver tissue. 1: GSK3β (phospho Ser9) Polyclonal Antibody was diluted at 1:200 (4 degree Celsius,overnight). 2: Sodium citrate pH 6.0 was used for antibody retrieval (>98 degree Celsius,20min). 3: Secondary antibody was diluted at 1:200 (room temperature, 30min). Negative control was used by secondary antibody only. Report Number 90284-l Host Rabbit Application IHC-P Clonality Polyclonal Model Number STJ90284 Clone ID NA Antibody Name Anti-Phospho-GSK3β (S9) antibody Testing Species RAT Testing Tissue LIVER ANTIBODY VALIDATION REPORT a. (A small amount of distilled water was added into the incubation box to prevent evaporation of antibody). 128. Secondary antibody incubation a. Slides were washed 3 times, with PBS on a shaker for 5min. Shortly after the slides were dried the corresponding secondary antibody solution was added (HRP labelled), covering the tissues, and incubated at room temperature for 30min. b. 129. DAB staining a. Slides were washed 3 times, with PBS on a shaker for 5min. b. Shortly after, the slides were dried and fresh DAB staining buffer was added inside the circles. The staining time was adjusted under a microscope. Yellow-brown colour represented a positive result. Slides were washed with water to stop the staining. c. 130. Haematoxylin staining a. Haematoxylin was used to counter-staining for 1min, and then the slides were washed with water. 1% Hydrochloric acid and alcohol was added for several seconds and then washed with water. Ammonia was used to reveal blue colour, and then flushed with water. b. 131. Desolation and Clearing i. Slides were incubated sequentially into: 75% alcohol 5min, 85% alcohol 5min, Anhydrous ethanol - 5min, Anhydrous ethanol - 5min & Xylene - 5min. Shortly after slides were dried and neutral gum was used to seal the slides. ii. 132. Visualization a. Results were validated with microscope, and the slides were scanned. Paraffin-Embedded Immunohistochemistry Protocol 122. 123. Tissue processing a. Slides were incubated sequentially into Xylene; 15min – Xylene, 15min - Anhydrous ethanol, 5min - Anhydrous ethanol, 5min - 85% alcohol, 5min - 75% alcohol & 5min – wash in distilled water. b. 124. Antigen retrieval a. Tissue slides were incubated with citric acid (PH6.0) antigen retrieval buffer and microwaved for antigen retrieval (heated until boiled and then stopped heating) for 8min. Slides were then heated with medium power for 7min. During this process slides were kept from drying out. After cooling down at room temperature, slides were washed with PBS on shaker for 5min, repeated for 3 times. b. 125. Inhibition of endogenous peroxidase a. Slides were placed in 3% Hydrogen peroxide solution, and incubated for 10 min at room temperature without light exposure. Slides were then washed 3 times with PBS on a shaker for 5mins. b. 126. BSA Blocking a. Shortly after slides were dried, a PAP pen was used to draw circles around the tissue sections (and to prevent draining of the antibody solution). Inside the circles, BSA was used to cover the tissue evenly, blocking for 30min. b. 127. Primary antibody incubation After blocking solution was removed a 1:200 solution of primary antibody/PBS was added on the slide, and incubated overnight at 4°C. St John's Laboratory Ltd. www.stjohnslabs.com
  • 13. Figure: Immunohistochemical analysis of paraffin embedded Rat lung tissue. 1: GSK3β (phospho Ser9) Polyclonal Antibody was diluted at 1:200 (4 degree Celsius,overnight). 2: Sodium citrate pH 6.0 was used for antibody retrieval (>98 degree Celsius,20min). 3: Secondary antibody was diluted at 1:200 (room temperature, 30min). Negative control was used by secondary antibody only. Report Number 90284-m Host Rabbit Application IHC-P Clonality Polyclonal Model Number STJ90284 Clone ID NA Antibody Name Anti-Phospho-GSK3β (S9) antibody Testing Species RAT Testing Tissue LUNG ANTIBODY VALIDATION REPORT a. (A small amount of distilled water was added into the incubation box to prevent evaporation of antibody). 117. Secondary antibody incubation a. Slides were washed 3 times, with PBS on a shaker for 5min. Shortly after the slides were dried the corresponding secondary antibody solution was added (HRP labelled), covering the tissues, and incubated at room temperature for 30min. b. 118. DAB staining a. Slides were washed 3 times, with PBS on a shaker for 5min. b. Shortly after, the slides were dried and fresh DAB staining buffer was added inside the circles. The staining time was adjusted under a microscope. Yellow-brown colour represented a positive result. Slides were washed with water to stop the staining. c. 119. Haematoxylin staining a. Haematoxylin was used to counter-staining for 1min, and then the slides were washed with water. 1% Hydrochloric acid and alcohol was added for several seconds and then washed with water. Ammonia was used to reveal blue colour, and then flushed with water. b. 120. Desolation and Clearing i. Slides were incubated sequentially into: 75% alcohol 5min, 85% alcohol 5min, Anhydrous ethanol - 5min, Anhydrous ethanol - 5min & Xylene - 5min. Shortly after slides were dried and neutral gum was used to seal the slides. ii. 121. Visualization a. Results were validated with microscope, and the slides were scanned. Paraffin-Embedded Immunohistochemistry Protocol 111. 112. Tissue processing a. Slides were incubated sequentially into Xylene; 15min – Xylene, 15min - Anhydrous ethanol, 5min - Anhydrous ethanol, 5min - 85% alcohol, 5min - 75% alcohol & 5min – wash in distilled water. b. 113. Antigen retrieval a. Tissue slides were incubated with citric acid (PH6.0) antigen retrieval buffer and microwaved for antigen retrieval (heated until boiled and then stopped heating) for 8min. Slides were then heated with medium power for 7min. During this process slides were kept from drying out. After cooling down at room temperature, slides were washed with PBS on shaker for 5min, repeated for 3 times. b. 114. Inhibition of endogenous peroxidase a. Slides were placed in 3% Hydrogen peroxide solution, and incubated for 10 min at room temperature without light exposure. Slides were then washed 3 times with PBS on a shaker for 5mins. b. 115. BSA Blocking a. Shortly after slides were dried, a PAP pen was used to draw circles around the tissue sections (and to prevent draining of the antibody solution). Inside the circles, BSA was used to cover the tissue evenly, blocking for 30min. b. 116. Primary antibody incubation After blocking solution was removed a 1:200 solution of primary antibody/PBS was added on the slide, and incubated overnight at 4°C. St John's Laboratory Ltd. www.stjohnslabs.com
  • 14. Figure: Immunohistochemical analysis of paraffin embedded Rat kidney tissue. 1: GSK3β (phospho Ser9) Polyclonal Antibody was diluted at 1:200 (4 degree Celsius,overnight). 2: Sodium citrate pH 6.0 was used for antibody retrieval (>98 degree Celsius,20min). 3: Secondary antibody was diluted at 1:200 (room temperature, 30min). Negative control was used by secondary antibody only. Report Number 90284-n Host Rabbit Application IHC-P Clonality Polyclonal Model Number STJ90284 Clone ID NA Antibody Name Anti-Phospho-GSK3β (S9) antibody Testing Species RAT Testing Tissue KIDNEY ANTIBODY VALIDATION REPORT a. (A small amount of distilled water was added into the incubation box to prevent evaporation of antibody). 106. Secondary antibody incubation a. Slides were washed 3 times, with PBS on a shaker for 5min. Shortly after the slides were dried the corresponding secondary antibody solution was added (HRP labelled), covering the tissues, and incubated at room temperature for 30min. b. 107. DAB staining a. Slides were washed 3 times, with PBS on a shaker for 5min. b. Shortly after, the slides were dried and fresh DAB staining buffer was added inside the circles. The staining time was adjusted under a microscope. Yellow-brown colour represented a positive result. Slides were washed with water to stop the staining. c. 108. Haematoxylin staining a. Haematoxylin was used to counter-staining for 1min, and then the slides were washed with water. 1% Hydrochloric acid and alcohol was added for several seconds and then washed with water. Ammonia was used to reveal blue colour, and then flushed with water. b. 109. Desolation and Clearing i. Slides were incubated sequentially into: 75% alcohol 5min, 85% alcohol 5min, Anhydrous ethanol - 5min, Anhydrous ethanol - 5min & Xylene - 5min. Shortly after slides were dried and neutral gum was used to seal the slides. ii. 110. Visualization a. Results were validated with microscope, and the slides were scanned. Paraffin-Embedded Immunohistochemistry Protocol 100. 101. Tissue processing a. Slides were incubated sequentially into Xylene; 15min – Xylene, 15min - Anhydrous ethanol, 5min - Anhydrous ethanol, 5min - 85% alcohol, 5min - 75% alcohol & 5min – wash in distilled water. b. 102. Antigen retrieval a. Tissue slides were incubated with citric acid (PH6.0) antigen retrieval buffer and microwaved for antigen retrieval (heated until boiled and then stopped heating) for 8min. Slides were then heated with medium power for 7min. During this process slides were kept from drying out. After cooling down at room temperature, slides were washed with PBS on shaker for 5min, repeated for 3 times. b. 103. Inhibition of endogenous peroxidase a. Slides were placed in 3% Hydrogen peroxide solution, and incubated for 10 min at room temperature without light exposure. Slides were then washed 3 times with PBS on a shaker for 5mins. b. 104. BSA Blocking a. Shortly after slides were dried, a PAP pen was used to draw circles around the tissue sections (and to prevent draining of the antibody solution). Inside the circles, BSA was used to cover the tissue evenly, blocking for 30min. b. 105. Primary antibody incubation After blocking solution was removed a 1:200 solution of primary antibody/PBS was added on the slide, and incubated overnight at 4°C. St John's Laboratory Ltd. www.stjohnslabs.com
  • 15. Figure: Immunohistochemical analysis of paraffin embedded Rat spinal cord tissue. 1: GSK3β (phospho Ser9) Polyclonal Antibody was diluted at 1:200 (4 degree Celsius,overnight). 2: Sodium citrate pH 6.0 was used for antibody retrieval (>98 degree Celsius,20min). 3: Secondary antibody was diluted at 1:200 (room temperature, 30min). Negative control was used by secondary antibody only. Report Number 90284-o Host Rabbit Application IHC-P Clonality Polyclonal Model Number STJ90284 Clone ID NA Antibody Name Anti-Phospho-GSK3β (S9) antibody Testing Species RAT Testing Tissue SPINAL ANTIBODY VALIDATION REPORT a. (A small amount of distilled water was added into the incubation box to prevent evaporation of antibody). 95. Secondary antibody incubation a. Slides were washed 3 times, with PBS on a shaker for 5min. Shortly after the slides were dried the corresponding secondary antibody solution was added (HRP labelled), covering the tissues, and incubated at room temperature for 30min. b. 96. DAB staining a. Slides were washed 3 times, with PBS on a shaker for 5min. b. Shortly after, the slides were dried and fresh DAB staining buffer was added inside the circles. The staining time was adjusted under a microscope. Yellow-brown colour represented a positive result. Slides were washed with water to stop the staining. c. 97. Haematoxylin staining a. Haematoxylin was used to counter-staining for 1min, and then the slides were washed with water. 1% Hydrochloric acid and alcohol was added for several seconds and then washed with water. Ammonia was used to reveal blue colour, and then flushed with water. b. 98. Desolation and Clearing i. Slides were incubated sequentially into: 75% alcohol 5min, 85% alcohol 5min, Anhydrous ethanol - 5min, Anhydrous ethanol - 5min & Xylene - 5min. Shortly after slides were dried and neutral gum was used to seal the slides. ii. 99. Visualization a. Results were validated with microscope, and the slides were scanned. Paraffin-Embedded Immunohistochemistry Protocol 89. 90. Tissue processing a. Slides were incubated sequentially into Xylene; 15min – Xylene, 15min - Anhydrous ethanol, 5min - Anhydrous ethanol, 5min - 85% alcohol, 5min - 75% alcohol & 5min – wash in distilled water. b. 91. Antigen retrieval a. Tissue slides were incubated with citric acid (PH6.0) antigen retrieval buffer and microwaved for antigen retrieval (heated until boiled and then stopped heating) for 8min. Slides were then heated with medium power for 7min. During this process slides were kept from drying out. After cooling down at room temperature, slides were washed with PBS on shaker for 5min, repeated for 3 times. b. 92. Inhibition of endogenous peroxidase a. Slides were placed in 3% Hydrogen peroxide solution, and incubated for 10 min at room temperature without light exposure. Slides were then washed 3 times with PBS on a shaker for 5mins. b. 93. BSA Blocking a. Shortly after slides were dried, a PAP pen was used to draw circles around the tissue sections (and to prevent draining of the antibody solution). Inside the circles, BSA was used to cover the tissue evenly, blocking for 30min. b. 94. Primary antibody incubation After blocking solution was removed a 1:200 solution of primary antibody/PBS was added on the slide, and incubated overnight at 4°C. St John's Laboratory Ltd. www.stjohnslabs.com
  • 16. Figure: Immunohistochemical analysis of paraffin embedded Rat brain tissue. 1: GSK3β (phospho Ser9) Polyclonal Antibody was diluted at 1:200 (4 degree Celsius,overnight). 2: Sodium citrate pH 6.0 was used for antibody retrieval (>98 degree Celsius,20min). 3: Secondary antibody was diluted at 1:200 (room temperature, 30min). Negative control was used by secondary antibody only. Report Number 90284-p Host Rabbit Application IHC-P Clonality Polyclonal Model Number STJ90284 Clone ID NA Antibody Name Anti-Phospho-GSK3β (S9) antibody Testing Species RAT Testing Tissue BRAIN ANTIBODY VALIDATION REPORT a. (A small amount of distilled water was added into the incubation box to prevent evaporation of antibody). 84. Secondary antibody incubation a. Slides were washed 3 times, with PBS on a shaker for 5min. Shortly after the slides were dried the corresponding secondary antibody solution was added (HRP labelled), covering the tissues, and incubated at room temperature for 30min. b. 85. DAB staining a. Slides were washed 3 times, with PBS on a shaker for 5min. b. Shortly after, the slides were dried and fresh DAB staining buffer was added inside the circles. The staining time was adjusted under a microscope. Yellow-brown colour represented a positive result. Slides were washed with water to stop the staining. c. 86. Haematoxylin staining a. Haematoxylin was used to counter-staining for 1min, and then the slides were washed with water. 1% Hydrochloric acid and alcohol was added for several seconds and then washed with water. Ammonia was used to reveal blue colour, and then flushed with water. b. 87. Desolation and Clearing i. Slides were incubated sequentially into: 75% alcohol 5min, 85% alcohol 5min, Anhydrous ethanol - 5min, Anhydrous ethanol - 5min & Xylene - 5min. Shortly after slides were dried and neutral gum was used to seal the slides. ii. 88. Visualization a. Results were validated with microscope, and the slides were scanned. Paraffin-Embedded Immunohistochemistry Protocol 78. 79. Tissue processing a. Slides were incubated sequentially into Xylene; 15min – Xylene, 15min - Anhydrous ethanol, 5min - Anhydrous ethanol, 5min - 85% alcohol, 5min - 75% alcohol & 5min – wash in distilled water. b. 80. Antigen retrieval a. Tissue slides were incubated with citric acid (PH6.0) antigen retrieval buffer and microwaved for antigen retrieval (heated until boiled and then stopped heating) for 8min. Slides were then heated with medium power for 7min. During this process slides were kept from drying out. After cooling down at room temperature, slides were washed with PBS on shaker for 5min, repeated for 3 times. b. 81. Inhibition of endogenous peroxidase a. Slides were placed in 3% Hydrogen peroxide solution, and incubated for 10 min at room temperature without light exposure. Slides were then washed 3 times with PBS on a shaker for 5mins. b. 82. BSA Blocking a. Shortly after slides were dried, a PAP pen was used to draw circles around the tissue sections (and to prevent draining of the antibody solution). Inside the circles, BSA was used to cover the tissue evenly, blocking for 30min. b. 83. Primary antibody incubation After blocking solution was removed a 1:200 solution of primary antibody/PBS was added on the slide, and incubated overnight at 4°C. St John's Laboratory Ltd. www.stjohnslabs.com
  • 17. Figure: Immunohistochemical analysis of paraffin embedded Rat spleen tissue. 1: GSK3β (phospho Ser9) Polyclonal Antibody was diluted at 1:200 (4 degree Celsius,overnight). 2: Sodium citrate pH 6.0 was used for antibody retrieval (>98 degree Celsius,20min). 3: Secondary antibody was diluted at 1:200 (room temperature, 30min). Negative control was used by secondary antibody only. Report Number 90284-q Host Rabbit Application IHC-P Clonality Polyclonal Model Number STJ90284 Clone ID NA Antibody Name Anti-Phospho-GSK3β (S9) antibody Testing Species RAT Testing Tissue SPLEEN ANTIBODY VALIDATION REPORT a. (A small amount of distilled water was added into the incubation box to prevent evaporation of antibody). 73. Secondary antibody incubation a. Slides were washed 3 times, with PBS on a shaker for 5min. Shortly after the slides were dried the corresponding secondary antibody solution was added (HRP labelled), covering the tissues, and incubated at room temperature for 30min. b. 74. DAB staining a. Slides were washed 3 times, with PBS on a shaker for 5min. b. Shortly after, the slides were dried and fresh DAB staining buffer was added inside the circles. The staining time was adjusted under a microscope. Yellow-brown colour represented a positive result. Slides were washed with water to stop the staining. c. 75. Haematoxylin staining a. Haematoxylin was used to counter-staining for 1min, and then the slides were washed with water. 1% Hydrochloric acid and alcohol was added for several seconds and then washed with water. Ammonia was used to reveal blue colour, and then flushed with water. b. 76. Desolation and Clearing i. Slides were incubated sequentially into: 75% alcohol 5min, 85% alcohol 5min, Anhydrous ethanol - 5min, Anhydrous ethanol - 5min & Xylene - 5min. Shortly after slides were dried and neutral gum was used to seal the slides. ii. 77. Visualization a. Results were validated with microscope, and the slides were scanned. Paraffin-Embedded Immunohistochemistry Protocol 67. 68. Tissue processing a. Slides were incubated sequentially into Xylene; 15min – Xylene, 15min - Anhydrous ethanol, 5min - Anhydrous ethanol, 5min - 85% alcohol, 5min - 75% alcohol & 5min – wash in distilled water. b. 69. Antigen retrieval a. Tissue slides were incubated with citric acid (PH6.0) antigen retrieval buffer and microwaved for antigen retrieval (heated until boiled and then stopped heating) for 8min. Slides were then heated with medium power for 7min. During this process slides were kept from drying out. After cooling down at room temperature, slides were washed with PBS on shaker for 5min, repeated for 3 times. b. 70. Inhibition of endogenous peroxidase a. Slides were placed in 3% Hydrogen peroxide solution, and incubated for 10 min at room temperature without light exposure. Slides were then washed 3 times with PBS on a shaker for 5mins. b. 71. BSA Blocking a. Shortly after slides were dried, a PAP pen was used to draw circles around the tissue sections (and to prevent draining of the antibody solution). Inside the circles, BSA was used to cover the tissue evenly, blocking for 30min. b. 72. Primary antibody incubation After blocking solution was removed a 1:200 solution of primary antibody/PBS was added on the slide, and incubated overnight at 4°C. St John's Laboratory Ltd. www.stjohnslabs.com
  • 18. Figure: Immunohistochemical analysis of paraffin embedded Mouse heart tissue. 1: GSK3β (phospho Ser9) Polyclonal Antibody was diluted at 1:200 (4 degree Celsius,overnight). 2: Sodium citrate pH 6.0 was used for antibody retrieval (>98 degree Celsius,20min). 3: Secondary antibody was diluted at 1:200 (room temperature, 30min). Negative control was used by secondary antibody only. Report Number 90284-r Host Rabbit Application IHC-P Clonality Polyclonal Model Number STJ90284 Clone ID NA Antibody Name Anti-Phospho-GSK3β (S9) antibody Testing Species MOUSE Testing Tissue HEART ANTIBODY VALIDATION REPORT a. (A small amount of distilled water was added into the incubation box to prevent evaporation of antibody). 62. Secondary antibody incubation a. Slides were washed 3 times, with PBS on a shaker for 5min. Shortly after the slides were dried the corresponding secondary antibody solution was added (HRP labelled), covering the tissues, and incubated at room temperature for 30min. b. 63. DAB staining a. Slides were washed 3 times, with PBS on a shaker for 5min. b. Shortly after, the slides were dried and fresh DAB staining buffer was added inside the circles. The staining time was adjusted under a microscope. Yellow-brown colour represented a positive result. Slides were washed with water to stop the staining. c. 64. Haematoxylin staining a. Haematoxylin was used to counter-staining for 1min, and then the slides were washed with water. 1% Hydrochloric acid and alcohol was added for several seconds and then washed with water. Ammonia was used to reveal blue colour, and then flushed with water. b. 65. Desolation and Clearing i. Slides were incubated sequentially into: 75% alcohol 5min, 85% alcohol 5min, Anhydrous ethanol - 5min, Anhydrous ethanol - 5min & Xylene - 5min. Shortly after slides were dried and neutral gum was used to seal the slides. ii. 66. Visualization a. Results were validated with microscope, and the slides were scanned. Paraffin-Embedded Immunohistochemistry Protocol 56. 57. Tissue processing a. Slides were incubated sequentially into Xylene; 15min – Xylene, 15min - Anhydrous ethanol, 5min - Anhydrous ethanol, 5min - 85% alcohol, 5min - 75% alcohol & 5min – wash in distilled water. b. 58. Antigen retrieval a. Tissue slides were incubated with citric acid (PH6.0) antigen retrieval buffer and microwaved for antigen retrieval (heated until boiled and then stopped heating) for 8min. Slides were then heated with medium power for 7min. During this process slides were kept from drying out. After cooling down at room temperature, slides were washed with PBS on shaker for 5min, repeated for 3 times. b. 59. Inhibition of endogenous peroxidase a. Slides were placed in 3% Hydrogen peroxide solution, and incubated for 10 min at room temperature without light exposure. Slides were then washed 3 times with PBS on a shaker for 5mins. b. 60. BSA Blocking a. Shortly after slides were dried, a PAP pen was used to draw circles around the tissue sections (and to prevent draining of the antibody solution). Inside the circles, BSA was used to cover the tissue evenly, blocking for 30min. b. 61. Primary antibody incubation After blocking solution was removed a 1:200 solution of primary antibody/PBS was added on the slide, and incubated overnight at 4°C. St John's Laboratory Ltd. www.stjohnslabs.com
  • 19. Figure: Immunohistochemical analysis of paraffin embedded Mouse testis tissue. 1: GSK3β (phospho Ser9) Polyclonal Antibody was diluted at 1:200 (4 degree Celsius,overnight). 2: Sodium citrate pH 6.0 was used for antibody retrieval (>98 degree Celsius,20min). 3: Secondary antibody was diluted at 1:200 (room temperature, 30min). Negative control was used by secondary antibody only. Report Number 90284-s Host Rabbit Application IHC-P Clonality Polyclonal Model Number STJ90284 Clone ID NA Antibody Name Anti-Phospho-GSK3β (S9) antibody Testing Species MOUSE Testing Tissue TESTIS ANTIBODY VALIDATION REPORT a. (A small amount of distilled water was added into the incubation box to prevent evaporation of antibody). 51. Secondary antibody incubation a. Slides were washed 3 times, with PBS on a shaker for 5min. Shortly after the slides were dried the corresponding secondary antibody solution was added (HRP labelled), covering the tissues, and incubated at room temperature for 30min. b. 52. DAB staining a. Slides were washed 3 times, with PBS on a shaker for 5min. b. Shortly after, the slides were dried and fresh DAB staining buffer was added inside the circles. The staining time was adjusted under a microscope. Yellow-brown colour represented a positive result. Slides were washed with water to stop the staining. c. 53. Haematoxylin staining a. Haematoxylin was used to counter-staining for 1min, and then the slides were washed with water. 1% Hydrochloric acid and alcohol was added for several seconds and then washed with water. Ammonia was used to reveal blue colour, and then flushed with water. b. 54. Desolation and Clearing i. Slides were incubated sequentially into: 75% alcohol 5min, 85% alcohol 5min, Anhydrous ethanol - 5min, Anhydrous ethanol - 5min & Xylene - 5min. Shortly after slides were dried and neutral gum was used to seal the slides. ii. 55. Visualization a. Results were validated with microscope, and the slides were scanned. Paraffin-Embedded Immunohistochemistry Protocol 45. 46. Tissue processing a. Slides were incubated sequentially into Xylene; 15min – Xylene, 15min - Anhydrous ethanol, 5min - Anhydrous ethanol, 5min - 85% alcohol, 5min - 75% alcohol & 5min – wash in distilled water. b. 47. Antigen retrieval a. Tissue slides were incubated with citric acid (PH6.0) antigen retrieval buffer and microwaved for antigen retrieval (heated until boiled and then stopped heating) for 8min. Slides were then heated with medium power for 7min. During this process slides were kept from drying out. After cooling down at room temperature, slides were washed with PBS on shaker for 5min, repeated for 3 times. b. 48. Inhibition of endogenous peroxidase a. Slides were placed in 3% Hydrogen peroxide solution, and incubated for 10 min at room temperature without light exposure. Slides were then washed 3 times with PBS on a shaker for 5mins. b. 49. BSA Blocking a. Shortly after slides were dried, a PAP pen was used to draw circles around the tissue sections (and to prevent draining of the antibody solution). Inside the circles, BSA was used to cover the tissue evenly, blocking for 30min. b. 50. Primary antibody incubation After blocking solution was removed a 1:200 solution of primary antibody/PBS was added on the slide, and incubated overnight at 4°C. St John's Laboratory Ltd. www.stjohnslabs.com
  • 20. Figure: Immunohistochemical analysis of paraffin embedded Mouse liver tissue. 1: GSK3β (phospho Ser9) Polyclonal Antibody was diluted at 1:200 (4 degree Celsius,overnight). 2: Sodium citrate pH 6.0 was used for antibody retrieval (>98 degree Celsius,20min). 3: Secondary antibody was diluted at 1:200 (room temperature, 30min). Negative control was used by secondary antibody only. Report Number 90284-t Host Rabbit Application IHC-P Clonality Polyclonal Model Number STJ90284 Clone ID NA Antibody Name Anti-Phospho-GSK3β (S9) antibody Testing Species MOUSE Testing Tissue LIVER ANTIBODY VALIDATION REPORT a. (A small amount of distilled water was added into the incubation box to prevent evaporation of antibody). 40. Secondary antibody incubation a. Slides were washed 3 times, with PBS on a shaker for 5min. Shortly after the slides were dried the corresponding secondary antibody solution was added (HRP labelled), covering the tissues, and incubated at room temperature for 30min. b. 41. DAB staining a. Slides were washed 3 times, with PBS on a shaker for 5min. b. Shortly after, the slides were dried and fresh DAB staining buffer was added inside the circles. The staining time was adjusted under a microscope. Yellow-brown colour represented a positive result. Slides were washed with water to stop the staining. c. 42. Haematoxylin staining a. Haematoxylin was used to counter-staining for 1min, and then the slides were washed with water. 1% Hydrochloric acid and alcohol was added for several seconds and then washed with water. Ammonia was used to reveal blue colour, and then flushed with water. b. 43. Desolation and Clearing i. Slides were incubated sequentially into: 75% alcohol 5min, 85% alcohol 5min, Anhydrous ethanol - 5min, Anhydrous ethanol - 5min & Xylene - 5min. Shortly after slides were dried and neutral gum was used to seal the slides. ii. 44. Visualization a. Results were validated with microscope, and the slides were scanned. Paraffin-Embedded Immunohistochemistry Protocol 34. 35. Tissue processing a. Slides were incubated sequentially into Xylene; 15min – Xylene, 15min - Anhydrous ethanol, 5min - Anhydrous ethanol, 5min - 85% alcohol, 5min - 75% alcohol & 5min – wash in distilled water. b. 36. Antigen retrieval a. Tissue slides were incubated with citric acid (PH6.0) antigen retrieval buffer and microwaved for antigen retrieval (heated until boiled and then stopped heating) for 8min. Slides were then heated with medium power for 7min. During this process slides were kept from drying out. After cooling down at room temperature, slides were washed with PBS on shaker for 5min, repeated for 3 times. b. 37. Inhibition of endogenous peroxidase a. Slides were placed in 3% Hydrogen peroxide solution, and incubated for 10 min at room temperature without light exposure. Slides were then washed 3 times with PBS on a shaker for 5mins. b. 38. BSA Blocking a. Shortly after slides were dried, a PAP pen was used to draw circles around the tissue sections (and to prevent draining of the antibody solution). Inside the circles, BSA was used to cover the tissue evenly, blocking for 30min. b. 39. Primary antibody incubation After blocking solution was removed a 1:200 solution of primary antibody/PBS was added on the slide, and incubated overnight at 4°C. St John's Laboratory Ltd. www.stjohnslabs.com
  • 21. Figure: Immunohistochemical analysis of paraffin embedded Mouse colon tissue. 1: GSK3β (phospho Ser9) Polyclonal Antibody was diluted at 1:200 (4 degree Celsius,overnight). 2: Sodium citrate pH 6.0 was used for antibody retrieval (>98 degree Celsius,20min). 3: Secondary antibody was diluted at 1:200 (room temperature, 30min). Negative control was used by secondary antibody only. Report Number 90284-u Host Rabbit Application IHC-P Clonality Polyclonal Model Number STJ90284 Clone ID NA Antibody Name Anti-Phospho-GSK3β (S9) antibody Testing Species MOUSE Testing Tissue COLON ANTIBODY VALIDATION REPORT a. (A small amount of distilled water was added into the incubation box to prevent evaporation of antibody). 29. Secondary antibody incubation a. Slides were washed 3 times, with PBS on a shaker for 5min. Shortly after the slides were dried the corresponding secondary antibody solution was added (HRP labelled), covering the tissues, and incubated at room temperature for 30min. b. 30. DAB staining a. Slides were washed 3 times, with PBS on a shaker for 5min. b. Shortly after, the slides were dried and fresh DAB staining buffer was added inside the circles. The staining time was adjusted under a microscope. Yellow-brown colour represented a positive result. Slides were washed with water to stop the staining. c. 31. Haematoxylin staining a. Haematoxylin was used to counter-staining for 1min, and then the slides were washed with water. 1% Hydrochloric acid and alcohol was added for several seconds and then washed with water. Ammonia was used to reveal blue colour, and then flushed with water. b. 32. Desolation and Clearing i. Slides were incubated sequentially into: 75% alcohol 5min, 85% alcohol 5min, Anhydrous ethanol - 5min, Anhydrous ethanol - 5min & Xylene - 5min. Shortly after slides were dried and neutral gum was used to seal the slides. ii. 33. Visualization a. Results were validated with microscope, and the slides were scanned. Paraffin-Embedded Immunohistochemistry Protocol 23. 24. Tissue processing a. Slides were incubated sequentially into Xylene; 15min – Xylene, 15min - Anhydrous ethanol, 5min - Anhydrous ethanol, 5min - 85% alcohol, 5min - 75% alcohol & 5min – wash in distilled water. b. 25. Antigen retrieval a. Tissue slides were incubated with citric acid (PH6.0) antigen retrieval buffer and microwaved for antigen retrieval (heated until boiled and then stopped heating) for 8min. Slides were then heated with medium power for 7min. During this process slides were kept from drying out. After cooling down at room temperature, slides were washed with PBS on shaker for 5min, repeated for 3 times. b. 26. Inhibition of endogenous peroxidase a. Slides were placed in 3% Hydrogen peroxide solution, and incubated for 10 min at room temperature without light exposure. Slides were then washed 3 times with PBS on a shaker for 5mins. b. 27. BSA Blocking a. Shortly after slides were dried, a PAP pen was used to draw circles around the tissue sections (and to prevent draining of the antibody solution). Inside the circles, BSA was used to cover the tissue evenly, blocking for 30min. b. 28. Primary antibody incubation After blocking solution was removed a 1:200 solution of primary antibody/PBS was added on the slide, and incubated overnight at 4°C. St John's Laboratory Ltd. www.stjohnslabs.com
  • 22. Figure: Immunohistochemical analysis of paraffin embedded Mouse lung tissue. 1: GSK3β (phospho Ser9) Polyclonal Antibody was diluted at 1:200 (4 degree Celsius,overnight). 2: Sodium citrate pH 6.0 was used for antibody retrieval (>98 degree Celsius,20min). 3: Secondary antibody was diluted at 1:200 (room temperature, 30min). Negative control was used by secondary antibody only. Report Number 90284-v Host Rabbit Application IHC-P Clonality Polyclonal Model Number STJ90284 Clone ID NA Antibody Name Anti-Phospho-GSK3β (S9) antibody Testing Species MOUSE Testing Tissue LUNG ANTIBODY VALIDATION REPORT b. (A small amount of distilled water was added into the incubation box to prevent evaporation of antibody). 18. Secondary antibody incubation a. Slides were washed 3 times, with PBS on a shaker for 5min. Shortly after the slides were dried the corresponding secondary antibody solution was added (HRP labelled), covering the tissues, and incubated at room temperature for 30min. b. 19. DAB staining a. Slides were washed 3 times, with PBS on a shaker for 5min. b. Shortly after, the slides were dried and fresh DAB staining buffer was added inside the circles. The staining time was adjusted under a microscope. Yellow-brown colour represented a positive result. Slides were washed with water to stop the staining. c. 20. Haematoxylin staining a. Haematoxylin was used to counter-staining for 1min, and then the slides were washed with water. 1% Hydrochloric acid and alcohol was added for several seconds and then washed with water. Ammonia was used to reveal blue colour, and then flushed with water. b. 21. Desolation and Clearing i. Slides were incubated sequentially into: 75% alcohol 5min, 85% alcohol 5min, Anhydrous ethanol - 5min, Anhydrous ethanol - 5min & Xylene - 5min. Shortly after slides were dried and neutral gum was used to seal the slides. ii. 22. Visualization a. Results were validated with microscope, and the slides were scanned. Paraffin-Embedded Immunohistochemistry Protocol 12. 13. Tissue processing a. Slides were incubated sequentially into Xylene; 15min – Xylene, 15min - Anhydrous ethanol, 5min - Anhydrous ethanol, 5min - 85% alcohol, 5min - 75% alcohol & 5min – wash in distilled water. b. 14. Antigen retrieval a. Tissue slides were incubated with citric acid (PH6.0) antigen retrieval buffer and microwaved for antigen retrieval (heated until boiled and then stopped heating) for 8min. Slides were then heated with medium power for 7min. During this process slides were kept from drying out. After cooling down at room temperature, slides were washed with PBS on shaker for 5min, repeated for 3 times. b. 15. Inhibition of endogenous peroxidase a. Slides were placed in 3% Hydrogen peroxide solution, and incubated for 10 min at room temperature without light exposure. Slides were then washed 3 times with PBS on a shaker for 5mins. b. 16. BSA Blocking a. Shortly after slides were dried, a PAP pen was used to draw circles around the tissue sections (and to prevent draining of the antibody solution). Inside the circles, BSA was used to cover the tissue evenly, blocking for 30min. b. 17. Primary antibody incubation After blocking solution was removed a 1:200 solution of primary antibody/PBS was added on the slide, and incubated overnight at 4°C. St John's Laboratory Ltd. www.stjohnslabs.com
  • 23. Figure: Immunohistochemical analysis of paraffin embedded Mouse kidney tissue. 1: GSK3β (phospho Ser9) Polyclonal Antibody was diluted at 1:200 (4 degree Celsius,overnight). 2: Sodium citrate pH 6.0 was used for antibody retrieval (>98 degree Celsius,20min). 3: Secondary antibody was diluted at 1:200 (room temperature, 30min). Negative control was used by secondary antibody only. Report Number 90284-w Host Rabbit Application IHC-P Clonality Polyclonal Model Number STJ90284 Clone ID NA Antibody Name Anti-Phospho-GSK3β (S9) antibody Testing Species MOUSE Testing Tissue KIDNEY ANTIBODY VALIDATION REPORT b. (A small amount of distilled water was added into the incubation box to prevent evaporation of antibody). 7. Secondary antibody incubation a. Slides were washed 3 times, with PBS on a shaker for 5min. Shortly after the slides were dried the corresponding secondary antibody solution was added (HRP labelled), covering the tissues, and incubated at room temperature for 30min. b. 8. DAB staining a. Slides were washed 3 times, with PBS on a shaker for 5min. b. Shortly after, the slides were dried and fresh DAB staining buffer was added inside the circles. The staining time was adjusted under a microscope. Yellow-brown colour represented a positive result. Slides were washed with water to stop the staining. c. 9. Haematoxylin staining a. Haematoxylin was used to counter-staining for 1min, and then the slides were washed with water. 1% Hydrochloric acid and alcohol was added for several seconds and then washed with water. Ammonia was used to reveal blue colour, and then flushed with water. b. 10. Desolation and Clearing i. Slides were incubated sequentially into: 75% alcohol 5min, 85% alcohol 5min, Anhydrous ethanol - 5min, Anhydrous ethanol - 5min & Xylene - 5min. Shortly after slides were dried and neutral gum was used to seal the slides. ii. 11. Visualization a. Results were validated with microscope, and the slides were scanned. Paraffin-Embedded Immunohistochemistry Protocol 1. 2. Tissue processing a. Slides were incubated sequentially into Xylene; 15min – Xylene, 15min - Anhydrous ethanol, 5min - Anhydrous ethanol, 5min - 85% alcohol, 5min - 75% alcohol & 5min – wash in distilled water. b. 3. Antigen retrieval a. Tissue slides were incubated with citric acid (PH6.0) antigen retrieval buffer and microwaved for antigen retrieval (heated until boiled and then stopped heating) for 8min. Slides were then heated with medium power for 7min. During this process slides were kept from drying out. After cooling down at room temperature, slides were washed with PBS on shaker for 5min, repeated for 3 times. b. 4. Inhibition of endogenous peroxidase a. Slides were placed in 3% Hydrogen peroxide solution, and incubated for 10 min at room temperature without light exposure. Slides were then washed 3 times with PBS on a shaker for 5mins. b. 5. BSA Blocking a. Shortly after slides were dried, a PAP pen was used to draw circles around the tissue sections (and to prevent draining of the antibody solution). Inside the circles, BSA was used to cover the tissue evenly, blocking for 30min. b. 6. Primary antibody incubation After blocking solution was removed a 1:200 solution of primary antibody/PBS was added on the slide, and incubated overnight at 4°C. St John's Laboratory Ltd. www.stjohnslabs.com
  • 24. Figure: Immunohistochemical analysis of paraffin embedded Mouse brain tissue. 1: GSK3β (phospho Ser9) Polyclonal Antibody was diluted at 1:200 (4 degree Celsius,overnight). 2: Sodium citrate pH 6.0 was used for antibody retrieval (>98 degree Celsius,20min). 3: Secondary antibody was diluted at 1:200 (room temperature, 30min). Negative control was used by secondary antibody only. Report Number 90284-x Host Rabbit Application IHC-P Clonality Polyclonal Model Number STJ90284 Clone ID NA Antibody Name Anti-Phospho-GSK3β (S9) antibody Testing Species MOUSE Testing Tissue BRAIN ANTIBODY VALIDATION REPORT c. (A small amount of distilled water was added into the incubation box to prevent evaporation of antibody). 254. Secondary antibody incubation a. Slides were washed 3 times, with PBS on a shaker for 5min. Shortly after the slides were dried the corresponding secondary antibody solution was added (HRP labelled), covering the tissues, and incubated at room temperature for 30min. b. 255. DAB staining a. Slides were washed 3 times, with PBS on a shaker for 5min. b. Shortly after, the slides were dried and fresh DAB staining buffer was added inside the circles. The staining time was adjusted under a microscope. Yellow-brown colour represented a positive result. Slides were washed with water to stop the staining. c. 256. Haematoxylin staining a. Haematoxylin was used to counter-staining for 1min, and then the slides were washed with water. 1% Hydrochloric acid and alcohol was added for several seconds and then washed with water. Ammonia was used to reveal blue colour, and then flushed with water. b. 257. Desolation and Clearing i. Slides were incubated sequentially into: 75% alcohol 5min, 85% alcohol 5min, Anhydrous ethanol - 5min, Anhydrous ethanol - 5min & Xylene - 5min. Shortly after slides were dried and neutral gum was used to seal the slides. ii. 258. Visualization a. Results were validated with microscope, and the slides were scanned. Paraffin-Embedded Immunohistochemistry Protocol 259. 260. Tissue processing a. Slides were incubated sequentially into Xylene; 15min – Xylene, 15min - Anhydrous ethanol, 5min - Anhydrous ethanol, 5min - 85% alcohol, 5min - 75% alcohol & 5min – wash in distilled water. b. 261. Antigen retrieval a. Tissue slides were incubated with citric acid (PH6.0) antigen retrieval buffer and microwaved for antigen retrieval (heated until boiled and then stopped heating) for 8min. Slides were then heated with medium power for 7min. During this process slides were kept from drying out. After cooling down at room temperature, slides were washed with PBS on shaker for 5min, repeated for 3 times. b. 262. Inhibition of endogenous peroxidase a. Slides were placed in 3% Hydrogen peroxide solution, and incubated for 10 min at room temperature without light exposure. Slides were then washed 3 times with PBS on a shaker for 5mins. b. 263. BSA Blocking a. Shortly after slides were dried, a PAP pen was used to draw circles around the tissue sections (and to prevent draining of the antibody solution). Inside the circles, BSA was used to cover the tissue evenly, blocking for 30min. b. 264. Primary antibody incubation After blocking solution was removed a 1:200 solution of primary antibody/PBS was added on the slide, and incubated overnight at 4°C. St John's Laboratory Ltd. www.stjohnslabs.com
  • 25. Figure: Immunohistochemical analysis of paraffin embedded Mouse spleen tissue. 1: GSK3β (phospho Ser9) Polyclonal Antibody was diluted at 1:200 (4 degree Celsius,overnight). 2: Sodium citrate pH 6.0 was used for antibody retrieval (>98 degree Celsius,20min). 3: Secondary antibody was diluted at 1:200 (room temperature, 30min). Negative control was used by secondary antibody only. Report Number 90284-y Host Rabbit Application IHC-P Clonality Polyclonal Model Number STJ90284 Clone ID NA Antibody Name Anti-Phospho-GSK3β (S9) antibody Testing Species MOUSE Testing Tissue SPLEEN ANTIBODY VALIDATION REPORT b. (A small amount of distilled water was added into the incubation box to prevent evaporation of antibody). 265. Secondary antibody incubation a. Slides were washed 3 times, with PBS on a shaker for 5min. Shortly after the slides were dried the corresponding secondary antibody solution was added (HRP labelled), covering the tissues, and incubated at room temperature for 30min. b. 266. DAB staining a. Slides were washed 3 times, with PBS on a shaker for 5min. b. Shortly after, the slides were dried and fresh DAB staining buffer was added inside the circles. The staining time was adjusted under a microscope. Yellow-brown colour represented a positive result. Slides were washed with water to stop the staining. c. 267. Haematoxylin staining a. Haematoxylin was used to counter-staining for 1min, and then the slides were washed with water. 1% Hydrochloric acid and alcohol was added for several seconds and then washed with water. Ammonia was used to reveal blue colour, and then flushed with water. b. 268. Desolation and Clearing i. Slides were incubated sequentially into: 75% alcohol 5min, 85% alcohol 5min, Anhydrous ethanol - 5min, Anhydrous ethanol - 5min & Xylene - 5min. Shortly after slides were dried and neutral gum was used to seal the slides. ii. 269. Visualization a. Results were validated with microscope, and the slides were scanned. Paraffin-Embedded Immunohistochemistry Protocol 270. 271. Tissue processing a. Slides were incubated sequentially into Xylene; 15min – Xylene, 15min - Anhydrous ethanol, 5min - Anhydrous ethanol, 5min - 85% alcohol, 5min - 75% alcohol & 5min – wash in distilled water. b. 272. Antigen retrieval a. Tissue slides were incubated with citric acid (PH6.0) antigen retrieval buffer and microwaved for antigen retrieval (heated until boiled and then stopped heating) for 8min. Slides were then heated with medium power for 7min. During this process slides were kept from drying out. After cooling down at room temperature, slides were washed with PBS on shaker for 5min, repeated for 3 times. b. 273. Inhibition of endogenous peroxidase a. Slides were placed in 3% Hydrogen peroxide solution, and incubated for 10 min at room temperature without light exposure. Slides were then washed 3 times with PBS on a shaker for 5mins. b. 274. BSA Blocking a. Shortly after slides were dried, a PAP pen was used to draw circles around the tissue sections (and to prevent draining of the antibody solution). Inside the circles, BSA was used to cover the tissue evenly, blocking for 30min. b. 275. Primary antibody incubation After blocking solution was removed a 1:200 solution of primary antibody/PBS was added on the slide, and incubated overnight at 4°C. St John's Laboratory Ltd. www.stjohnslabs.com