12
Figure: Immunohistochemical analysis of paraffin embedded Human colon tissue. 1: β-Tubulin Monoclonal Antibody(5G3) 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 96932-a Host Mouse Application IHC-P Clonality Monoclonal Model Number STJ96932 Clone ID 5G3 Antibody Name Anti-β-Tubulin antibody Testing Species HUMAN Testing Tissue COLON ANTIBODY VALIDATION REPORT (A small amount of distilled water was added into the incubation box to prevent evaporation of antibody). Secondary antibody incubation 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. DAB staining Slides were washed 3 times, with PBS on a shaker for 5min. 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. Haematoxylin staining 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. Desolation and Clearing 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. Visualization Results were validated with microscope, and the slides were scanned. Paraffin-Embedded Immunohistochemistry Protocol Tissue processing 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. Antigen retrieval 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. Inhibition of endogenous peroxidase 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. BSA Blocking 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. 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

Immunohistochemistry Antibody Validation Report for Anti-β-Tubulin Antibody (STJ96932)

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Figure:

Immunohistochemical

analysis of paraffin

embedded Human colon

tissue. 1: β-Tubulin

Monoclonal

Antibody(5G3) 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 96932-a Host Mouse

Application IHC-P Clonality Monoclonal

Model Number STJ96932 Clone ID 5G3

Antibody Name Anti-β-Tubulin 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).

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 Human lung

cancer tissue. 1: β-

Tubulin Monoclonal

Antibody(5G3) 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 96932-b Host Mouse

Application IHC-P Clonality Monoclonal

Model Number STJ96932 Clone ID 5G3

Antibody Name Anti-β-Tubulin 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).

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 Human

appendix tissue. 1: β-

Tubulin Monoclonal

Antibody(5G3) 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 96932-c Host Mouse

Application IHC-P Clonality Monoclonal

Model Number STJ96932 Clone ID 5G3

Antibody Name Anti-β-Tubulin 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).

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 Rat heart

tissue. 1: β-Tubulin

Monoclonal

Antibody(5G3) 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 96932-d Host Mouse

Application IHC-P Clonality Monoclonal

Model Number STJ96932 Clone ID 5G3

Antibody Name Anti-β-Tubulin 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).

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 Rat liver

tissue. 1: β-Tubulin

Monoclonal

Antibody(5G3) 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 96932-e Host Mouse

Application IHC-P Clonality Monoclonal

Model Number STJ96932 Clone ID 5G3

Antibody Name Anti-β-Tubulin 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).

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 Rat lung

tissue. 1: β-Tubulin

Monoclonal

Antibody(5G3) 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 96932-f Host Mouse

Application IHC-P Clonality Monoclonal

Model Number STJ96932 Clone ID 5G3

Antibody Name Anti-β-Tubulin 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).

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 Rat spleen

tissue. 1: β-Tubulin

Monoclonal

Antibody(5G3) 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 96932-g Host Mouse

Application IHC-P Clonality Monoclonal

Model Number STJ96932 Clone ID 5G3

Antibody Name Anti-β-Tubulin antibody

Testing Species RAT Testing Tissue SPLEEN

ANTIBODY VALIDATION REPORT

c. (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

Figure:

Immunohistochemical

analysis of paraffin

embedded Mouse testis

tissue. 1: β-Tubulin

Monoclonal

Antibody(5G3) 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 96932-h Host Mouse

Application IHC-P Clonality Monoclonal

Model Number STJ96932 Clone ID 5G3

Antibody Name Anti-β-Tubulin 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).

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.

St John's Laboratory Ltd. www.stjohnslabs.com

Figure:

Immunohistochemical

analysis of paraffin

embedded Mouse lung

tissue. 1: β-Tubulin

Monoclonal

Antibody(5G3) 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 96932-i Host Mouse

Application IHC-P Clonality Monoclonal

Model Number STJ96932 Clone ID 5G3

Antibody Name Anti-β-Tubulin 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).

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

Figure:

Immunohistochemical

analysis of paraffin

embedded Mouse

kidney tissue. 1: β-

Tubulin Monoclonal

Antibody(5G3) 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 96932-j Host Mouse

Application IHC-P Clonality Monoclonal

Model Number STJ96932 Clone ID 5G3

Antibody Name Anti-β-Tubulin 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).

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

Figure:

Immunohistochemical

analysis of paraffin

embedded Mouse

spleen tissue. 1: β-

Tubulin Monoclonal

Antibody(5G3) 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 96932-k Host Mouse

Application IHC-P Clonality Monoclonal

Model Number STJ96932 Clone ID 5G3

Antibody Name Anti-β-Tubulin 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).

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

Figure:

Immunohistochemical

analysis of paraffin

embedded Mouse colon

cancer tissue. 1: β-

Tubulin Monoclonal

Antibody(5G3) 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 96932-l Host Mouse

Application IHC-P Clonality Monoclonal

Model Number STJ96932 Clone ID 5G3

Antibody Name Anti-β-Tubulin antibody

Testing Species MOUSE Testing Tissue COLON CANCER

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