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Immunohistochemistry Antibody Validation Report for Anti-Phospho-Akt (S473) Antibody (STJ90166)

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RAC-alpha serine/threonine-protein kinase is an enzyme that in humans is encoded by the AKT1 gene. This enzyme belongs to the AKT subfamily of serine/threonine kinases that contain SH2 (Src homology 2-like) domains.The serine-threonine protein kinase AKT1 is catalytically inactive in serum-starved primary and immortalized fibroblasts. AKT1 and the related AKT2 are activated by platelet-derived growth factor. The activation is rapid and specific, and it is abrogated by mutations in the pleckstrin homology domain of AKT1.

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Immunohistochemistry Antibody Validation Report for Anti-Phospho-Akt (S473) Antibody (STJ90166)

  1. 1. Figure: Immunohistochemical analysis of paraffin embedded Human uterus tissue. 1: Akt (phospho Ser473) 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 90166-a Host Rabbit Application IHC-P Clonality Polyclonal Model Number STJ90166 Clone ID NA Antibody Name Anti-Phospho-Akt (S473) 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). 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
  2. 2. Figure: Immunohistochemical analysis of paraffin embedded Human uterus cancer tissue. 1: Akt (phospho Ser473) 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 90166-b Host Rabbit Application IHC-P Clonality Polyclonal Model Number STJ90166 Clone ID NA Antibody Name Anti-Phospho-Akt (S473) 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). 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
  3. 3. Figure: Immunohistochemical analysis of paraffin embedded Human Tonsil tissue. 1: Akt (phospho Ser473) 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 90166-c Host Rabbit Application IHC-P Clonality Polyclonal Model Number STJ90166 Clone ID NA Antibody Name Anti-Phospho-Akt (S473) antibody Testing Species HUMAN Testing Tissue TONSIL 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
  4. 4. Figure: Immunohistochemical analysis of paraffin embedded Human colon tissue. 1: Akt (phospho Ser473) 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 90166-d Host Rabbit Application IHC-P Clonality Polyclonal Model Number STJ90166 Clone ID NA Antibody Name Anti-Phospho-Akt (S473) 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). 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
  5. 5. Figure: Immunohistochemical analysis of paraffin embedded Human lung tissue. 1: Akt (phospho Ser473) 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 90166-e Host Rabbit Application IHC-P Clonality Polyclonal Model Number STJ90166 Clone ID NA Antibody Name Anti-Phospho-Akt (S473) 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). 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
  6. 6. Figure: Immunohistochemical analysis of paraffin embedded Human lung cancer tissue. 1: Akt (phospho Ser473) 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 90166-f Host Rabbit Application IHC-P Clonality Polyclonal Model Number STJ90166 Clone ID NA Antibody Name Anti-Phospho-Akt (S473) antibody Testing Species HUMAN Testing Tissue LUNG CANCER 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
  7. 7. Figure: Immunohistochemical analysis of paraffin embedded Human stomach tissue. 1: Akt (phospho Ser473) 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 90166-g Host Rabbit Application IHC-P Clonality Polyclonal Model Number STJ90166 Clone ID NA Antibody Name Anti-Phospho-Akt (S473) 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). 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
  8. 8. Figure: Immunohistochemical analysis of paraffin embedded Human appendix tissue. 1: Akt (phospho Ser473) 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 90166-h Host Rabbit Application IHC-P Clonality Polyclonal Model Number STJ90166 Clone ID NA Antibody Name Anti-Phospho-Akt (S473) 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). 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
  9. 9. Figure: Immunohistochemical analysis of paraffin embedded Rat testis tissue. 1: Akt (phospho Ser473) 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 90166-i Host Rabbit Application IHC-P Clonality Polyclonal Model Number STJ90166 Clone ID NA Antibody Name Anti-Phospho-Akt (S473) antibody Testing Species RAT Testing Tissue TESTIS ANTIBODY VALIDATION REPORT c. (A small amount of distilled water was added into the incubation box to prevent evaporation of antibody). 89. 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. 90. 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. 91. 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. 92. 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. 93. Visualization a. Results were validated with microscope, and the slides were scanned. Paraffin-Embedded Immunohistochemistry Protocol 94. 95. 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. 96. 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. 97. 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. 98. 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. 99. 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. 10. Figure: Immunohistochemical analysis of paraffin embedded Rat lung tissue. 1: Akt (phospho Ser473) 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 90166-j Host Rabbit Application IHC-P Clonality Polyclonal Model Number STJ90166 Clone ID NA Antibody Name Anti-Phospho-Akt (S473) antibody Testing Species RAT Testing Tissue LUNG ANTIBODY VALIDATION REPORT b. (A small amount of distilled water was added into the incubation box to prevent evaporation of antibody). 100. 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. 101. 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. 102. 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. 103. 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. 104. Visualization a. Results were validated with microscope, and the slides were scanned. Paraffin-Embedded Immunohistochemistry Protocol 105. 106. 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. 107. 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. 108. 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. 109. 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. 110. 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. 11. Figure: Immunohistochemical analysis of paraffin embedded Rat kidney tissue. 1: Akt (phospho Ser473) 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 90166-k Host Rabbit Application IHC-P Clonality Polyclonal Model Number STJ90166 Clone ID NA Antibody Name Anti-Phospho-Akt (S473) antibody Testing Species RAT Testing Tissue KIDNEY ANTIBODY VALIDATION REPORT b. (A small amount of distilled water was added into the incubation box to prevent evaporation of antibody). 111. 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. 112. 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. 113. 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. 114. 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. 115. Visualization a. Results were validated with microscope, and the slides were scanned. Paraffin-Embedded Immunohistochemistry Protocol 116. 117. 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. 118. 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. 119. 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. 120. 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. 121. 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. 12. Figure: Immunohistochemical analysis of paraffin embedded Rat spinal cord tissue. 1: Akt (phospho Ser473) 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 90166-l Host Rabbit Application IHC-P Clonality Polyclonal Model Number STJ90166 Clone ID NA Antibody Name Anti-Phospho-Akt (S473) antibody Testing Species RAT Testing Tissue SPINAL ANTIBODY VALIDATION REPORT b. (A small amount of distilled water was added into the incubation box to prevent evaporation of antibody). 122. 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. 123. 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. 124. 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. 125. 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. 126. Visualization a. Results were validated with microscope, and the slides were scanned. Paraffin-Embedded Immunohistochemistry Protocol 127. 128. 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. 129. 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. 130. 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. 131. 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. 132. 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. 13. Figure: Immunohistochemical analysis of paraffin embedded Rat brain tissue. 1: Akt (phospho Ser473) 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 90166-m Host Rabbit Application IHC-P Clonality Polyclonal Model Number STJ90166 Clone ID NA Antibody Name Anti-Phospho-Akt (S473) antibody Testing Species RAT Testing Tissue BRAIN ANTIBODY VALIDATION REPORT b. (A small amount of distilled water was added into the incubation box to prevent evaporation of antibody). 133. 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. 134. 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. 135. 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. 136. 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. 137. Visualization a. Results were validated with microscope, and the slides were scanned. Paraffin-Embedded Immunohistochemistry Protocol 138. 139. 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. 140. 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. 141. 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. 142. 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. 143. 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. 14. Figure: Immunohistochemical analysis of paraffin embedded Rat spleen tissue. 1: Akt (phospho Ser473) 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 90166-n Host Rabbit Application IHC-P Clonality Polyclonal Model Number STJ90166 Clone ID NA Antibody Name Anti-Phospho-Akt (S473) antibody Testing Species RAT Testing Tissue SPLEEN ANTIBODY VALIDATION REPORT b. (A small amount of distilled water was added into the incubation box to prevent evaporation of antibody). 144. 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. 145. 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. 146. 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. 147. 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. 148. Visualization a. Results were validated with microscope, and the slides were scanned. Paraffin-Embedded Immunohistochemistry Protocol 149. 150. 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. 151. 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. 152. 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. 153. 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. 154. 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. 15. Figure: Immunohistochemical analysis of paraffin embedded Mouse testis tissue. 1: Akt (phospho Ser473) 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 90166-o Host Rabbit Application IHC-P Clonality Polyclonal Model Number STJ90166 Clone ID NA Antibody Name Anti-Phospho-Akt (S473) antibody Testing Species MOUSE Testing Tissue TESTIS ANTIBODY VALIDATION REPORT b. (A small amount of distilled water was added into the incubation box to prevent evaporation of antibody). 155. 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. 156. 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. 157. 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. 158. 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. 159. Visualization a. Results were validated with microscope, and the slides were scanned. Paraffin-Embedded Immunohistochemistry Protocol 160. 161. 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. 162. 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. 163. 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. 164. 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. 165. 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. 16. Figure: Immunohistochemical analysis of paraffin embedded Mouse colon tissue. 1: Akt (phospho Ser473) 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 90166-p Host Rabbit Application IHC-P Clonality Polyclonal Model Number STJ90166 Clone ID NA Antibody Name Anti-Phospho-Akt (S473) antibody Testing Species MOUSE Testing Tissue COLON ANTIBODY VALIDATION REPORT b. (A small amount of distilled water was added into the incubation box to prevent evaporation of antibody). 166. 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. 167. 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. 168. 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. 169. 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. 170. Visualization a. Results were validated with microscope, and the slides were scanned. Paraffin-Embedded Immunohistochemistry Protocol 171. 172. 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. 173. 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. 174. 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. 175. 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. 176. 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. 17. Figure: Immunohistochemical analysis of paraffin embedded Mouse lung tissue. 1: Akt (phospho Ser473) 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 90166-q Host Rabbit Application IHC-P Clonality Polyclonal Model Number STJ90166 Clone ID NA Antibody Name Anti-Phospho-Akt (S473) 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). 177. 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. 178. 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. 179. 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. 180. 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. 181. Visualization a. Results were validated with microscope, and the slides were scanned. Paraffin-Embedded Immunohistochemistry Protocol 182. 183. 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. 184. 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. 185. 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. 186. 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. 187. 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. 18. Figure: Immunohistochemical analysis of paraffin embedded Mouse brain tissue. 1: Akt (phospho Ser473) 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 90166-r Host Rabbit Application IHC-P Clonality Polyclonal Model Number STJ90166 Clone ID NA Antibody Name Anti-Phospho-Akt (S473) antibody Testing Species MOUSE Testing Tissue BRAIN ANTIBODY VALIDATION REPORT b. (A small amount of distilled water was added into the incubation box to prevent evaporation of antibody). 188. 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. 189. 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. 190. 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. 191. 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. 192. Visualization a. Results were validated with microscope, and the slides were scanned. Paraffin-Embedded Immunohistochemistry Protocol 193. 194. 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. 195. 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. 196. 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. 197. 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. 198. 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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