Microtubules are assembled from dimers of α- and β-tubulin. These subunits are slightly acidic with an isoelectric point between 5.2 and 5.8. Each has a molecular weight of approximately 50 kDa. To form microtubules, the dimers of α- and β-tubulin bind to GTP and assemble onto the (+) ends of microtubules while in the GTP-bound state. The β-tubulin subunit is exposed on the plus end of the microtubule while the α-tubulin subunit is exposed on the minus end. After the dimer is incorporated into the microtubule, the molecule of GTP bound to the β-tubulin subunit eventually hydrolyzes into GDP through inter-dimer contacts along the microtubule protofilament. Whether the β-tubulin member of the tubulin dimer is bound to GTP or GDP influences the stability of the dimer in the microtubule. Dimers bound to GTP tend to assemble into microtubules, while dimers bound to GDP tend to fall apart; thus, this GTP cycle is essential for the dynamic instability of the microtubule. Anti-α-tubulin (Acetyl Lys40) -http://www.stjohnslabs.com/a-tubulin-acetyl-lys40-antibody-4a8 Join our Antibody Validation Project - http://www.stjohnslabs.com/services/antibody-validation

1. Figure:
Immunohistochemical
analysis of paraffin
embedded Human
uterus tissue. 1: α-
tubulin (Acetyl Lys40)
Monoclonal
Antibody(4A8) 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 97537-a Host Mouse
Application IHC-P Clonality Monoclonal
Model Number STJ97537 Clone ID 4A8
Antibody Name Anti-α-tubulin (Acetyl Lys40) 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).
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

2. Figure:
Immunohistochemical
analysis of paraffin
embedded Human liver
tissue. 1: α-tubulin
(Acetyl Lys40)
Monoclonal
Antibody(4A8) 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 97537-b Host Mouse
Application IHC-P Clonality Monoclonal
Model Number STJ97537 Clone ID 4A8
Antibody Name Anti-α-tubulin (Acetyl Lys40) 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).
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

3. Figure:
Immunohistochemical
analysis of paraffin
embedded Human liver
cancer tissue. 1: α-
tubulin (Acetyl Lys40)
Monoclonal
Antibody(4A8) 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 97537-c Host Mouse
Application IHC-P Clonality Monoclonal
Model Number STJ97537 Clone ID 4A8
Antibody Name Anti-α-tubulin (Acetyl Lys40) 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).
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

4. Figure:
Immunohistochemical
analysis of paraffin
embedded Human
stomach cancer tissue.
1: α-tubulin (Acetyl
Lys40) Monoclonal
Antibody(4A8) 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 97537-d Host Mouse
Application IHC-P Clonality Monoclonal
Model Number STJ97537 Clone ID 4A8
Antibody Name Anti-α-tubulin (Acetyl Lys40) 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).
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

5. Figure:
Immunohistochemical
analysis of paraffin
embedded Rat heart
tissue. 1: α-tubulin
(Acetyl Lys40)
Monoclonal
Antibody(4A8) 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 97537-e Host Mouse
Application IHC-P Clonality Monoclonal
Model Number STJ97537 Clone ID 4A8
Antibody Name Anti-α-tubulin (Acetyl Lys40) 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).
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

6. Figure:
Immunohistochemical
analysis of paraffin
embedded Rat kidney
tissue. 1: α-tubulin
(Acetyl Lys40)
Monoclonal
Antibody(4A8) 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 97537-f Host Mouse
Application IHC-P Clonality Monoclonal
Model Number STJ97537 Clone ID 4A8
Antibody Name Anti-α-tubulin (Acetyl Lys40) antibody
Testing Species RAT Testing Tissue KIDNEY
ANTIBODY VALIDATION REPORT
c. (A small amount of distilled water was added into the incubation
box to prevent evaporation of antibody).
56. 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.
57. 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.
58. 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.
59. 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.
60. Visualization
a. Results were validated with microscope, and the slides were
scanned.
Paraffin-Embedded
Immunohistochemistry Protocol
61.
62. 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.
63. 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.
64. 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.
65. 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.
66. 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 Mouse heart
tissue. 1: α-tubulin
(Acetyl Lys40)
Monoclonal
Antibody(4A8) 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 97537-g Host Mouse
Application IHC-P Clonality Monoclonal
Model Number STJ97537 Clone ID 4A8
Antibody Name Anti-α-tubulin (Acetyl Lys40) antibody
Testing Species MOUSE Testing Tissue HEART
ANTIBODY VALIDATION REPORT
b. (A small amount of distilled water was added into the incubation
box to prevent evaporation of antibody).
67. 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.
68. 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.
69. 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.
70. 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.
71. Visualization
a. Results were validated with microscope, and the slides were
scanned.
Paraffin-Embedded
Immunohistochemistry Protocol
72.
73. 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.
74. 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.
75. 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.
76. 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.
77. 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 Mouse
kidney tissue. 1: α-
tubulin (Acetyl Lys40)
Monoclonal
Antibody(4A8) 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 97537-h Host Mouse
Application IHC-P Clonality Monoclonal
Model Number STJ97537 Clone ID 4A8
Antibody Name Anti-α-tubulin (Acetyl Lys40) 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).
78. 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.
79. 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.
80. 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.
81. 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.
82. Visualization
a. Results were validated with microscope, and the slides were
scanned.
Paraffin-Embedded
Immunohistochemistry Protocol
83.
84. 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.
85. 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.
86. 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.
87. 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.
88. 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.
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