Figure:

Immunohistochemical

analysis of paraffin

embedded Human

uterus tissue. 1: HP-1α

Mouse Monoclonal

Antibody(5E3) 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 98895-a Host Mouse

Application IHC-P Clonality Monoclonal

Model Number STJ98895 Clone ID NA

Antibody Name Anti-HP-1α 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).

41. 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.

42. 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.

43. 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.

44. 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.

45. Visualization

a. Results were validated with microscope, and the slides were

scanned.

Paraffin-Embedded

Immunohistochemistry Protocol 35.

36. 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.

37. 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.

38. 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.

39. 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.

40. Primary antibody incubation

After blocking solution was removed a 1:200 solution of

primary antibody/PBS was added on the slide, and incubated

overnight at 4°C.

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

Figure:

Immunohistochemical

analysis of paraffin

embedded Human lung

tissue. 1: HP-1α Mouse

Monoclonal

Antibody(5E3) 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 98895-b Host Mouse

Application IHC-P Clonality Monoclonal

Model Number STJ98895 Clone ID NA

Antibody Name Anti-HP-1α 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).

30. 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.

31. 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.

32. 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.

33. 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.

34. Visualization

a. Results were validated with microscope, and the slides were

scanned.

Paraffin-Embedded

Immunohistochemistry Protocol 24.

25. 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.

26. 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.

27. 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.

28. 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.

29. 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: HP-1α Mouse

Monoclonal

Antibody(5E3) 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 98895-c Host Mouse

Application IHC-P Clonality Monoclonal

Model Number STJ98895 Clone ID NA

Antibody Name Anti-HP-1α 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).

19. 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.

20. 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.

21. 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.

22. 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.

23. Visualization

a. Results were validated with microscope, and the slides were

scanned.

Paraffin-Embedded

Immunohistochemistry Protocol 13.

14. 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.

15. 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.

16. 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.

17. 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.

18. 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: HP-1α Mouse

Monoclonal

Antibody(5E3) 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 98895-d Host Mouse

Application IHC-P Clonality Monoclonal

Model Number STJ98895 Clone ID NA

Antibody Name Anti-HP-1α antibody

Testing Species MOUSE Testing Tissue LUNG

ANTIBODY VALIDATION REPORT

a. (A small amount of distilled water was added into the incubation

box to prevent evaporation of antibody).

8. 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.

9. 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.

10. 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.

11. 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.

12. Visualization

a. Results were validated with microscope, and the slides were

scanned.

Paraffin-Embedded

Immunohistochemistry Protocol 2.

3. 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.

4. 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.

5. 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.

6. 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.

7. 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