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Figure:
Immunohistochemical
analysis of paraffin
embedded Human liver
tissue. 1: Amyloid-β
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 98585-a Host Rabbit
Application IHC-P Clonality Polyclonal
Model Number STJ98585 Clone ID NA
Antibody Name Anti-Amyloid-β 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
Figure:
Immunohistochemical
analysis of paraffin
embedded Rat lung
tissue. 1: Amyloid-β
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 98585-b Host Rabbit
Application IHC-P Clonality Polyclonal
Model Number STJ98585 Clone ID NA
Antibody Name Anti-Amyloid-β 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).
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 kidney
tissue. 1: Amyloid-β
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 98585-c Host Rabbit
Application IHC-P Clonality Polyclonal
Model Number STJ98585 Clone ID NA
Antibody Name Anti-Amyloid-β 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).
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 spleen
tissue. 1: Amyloid-β
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 98585-d Host Rabbit
Application IHC-P Clonality Polyclonal
Model Number STJ98585 Clone ID NA
Antibody Name Anti-Amyloid-β 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).
7. Secondary antibody incubation
a. Slides were washed 3 times, with PBS on a shaker for 5min.
Shortly after the slides were dried the corresponding secondary
antibody solution was added (HRP labelled), covering the
tissues, and incubated at room temperature for 30min.
b.
8. DAB staining
a. Slides were washed 3 times, with PBS on a shaker for 5min.
b. Shortly after, the slides were dried and fresh DAB staining buffer
was added inside the circles. The staining time was adjusted
under a microscope. Yellow-brown colour represented a positive
result. Slides were washed with water to stop the staining.
c.
9. Haematoxylin staining
a. Haematoxylin was used to counter-staining for 1min, and then
the slides were washed with water. 1% Hydrochloric acid and
alcohol was added for several seconds and then washed with
water. Ammonia was used to reveal blue colour, and then
flushed with water.
b.
10. Desolation and Clearing
i. Slides were incubated sequentially into: 75% alcohol 5min, 85%
alcohol 5min, Anhydrous ethanol - 5min, Anhydrous ethanol -
5min & Xylene - 5min. Shortly after slides were dried and neutral
gum was used to seal the slides.
ii.
11. Visualization
a. Results were validated with microscope, and the slides were
scanned.
Paraffin-Embedded
Immunohistochemistry Protocol
1.
2. Tissue processing
a. Slides were incubated sequentially into Xylene; 15min –
Xylene, 15min - Anhydrous ethanol, 5min - Anhydrous
ethanol, 5min - 85% alcohol, 5min - 75% alcohol & 5min –
wash in distilled water.
b.
3. Antigen retrieval
a. Tissue slides were incubated with citric acid (PH6.0) antigen
retrieval buffer and microwaved for antigen retrieval (heated
until boiled and then stopped heating) for 8min. Slides were
then heated with medium power for 7min. During this
process slides were kept from drying out. After cooling down
at room temperature, slides were washed with PBS on
shaker for 5min, repeated for 3 times.
b.
4. Inhibition of endogenous peroxidase
a. Slides were placed in 3% Hydrogen peroxide solution, and
incubated for 10 min at room temperature without light
exposure. Slides were then washed 3 times with PBS on a
shaker for 5mins.
b.
5. BSA Blocking
a. Shortly after slides were dried, a PAP pen was used to draw
circles around the tissue sections (and to prevent draining of
the antibody solution). Inside the circles, BSA was used to
cover the tissue evenly, blocking for 30min.
b.
6. Primary antibody incubation
After blocking solution was removed a 1:200 solution of
primary antibody/PBS was added on the slide, and incubated
overnight at 4°C.
St John's Laboratory Ltd.
www.stjohnslabs.com
Figure:
Immunohistochemical
analysis of paraffin
embedded Mouse heart
tissue. 1: Amyloid-β
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 98585-e Host Rabbit
Application IHC-P Clonality Polyclonal
Model Number STJ98585 Clone ID NA
Antibody Name Anti-Amyloid-β antibody
Testing Species MOUSE Testing Tissue HEART
ANTIBODY VALIDATION REPORT
c. (A small amount of distilled water was added into the incubation
box to prevent evaporation of antibody).
45. 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.
46. 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.
47. 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.
48. 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.
49. Visualization
a. Results were validated with microscope, and the slides were
scanned.
Paraffin-Embedded
Immunohistochemistry Protocol
50.
51. 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.
52. 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.
53. 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.
54. 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.
55. Primary antibody incubation
After blocking solution was removed a 1:200 solution of
primary antibody/PBS was added on the slide, and incubated
overnight at 4°C.
St John's Laboratory Ltd.
www.stjohnslabs.com
Figure:
Immunohistochemical
analysis of paraffin
embedded Mouse liver
tissue. 1: Amyloid-β
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 98585-f Host Rabbit
Application IHC-P Clonality Polyclonal
Model Number STJ98585 Clone ID NA
Antibody Name Anti-Amyloid-β antibody
Testing Species MOUSE Testing Tissue LIVER
ANTIBODY VALIDATION REPORT
b. (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
Figure:
Immunohistochemical
analysis of paraffin
embedded Mouse
kidney tissue. 1:
Amyloid-β 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 98585-g Host Rabbit
Application IHC-P Clonality Polyclonal
Model Number STJ98585 Clone ID NA
Antibody Name Anti-Amyloid-β 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).
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

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  • 1. Figure: Immunohistochemical analysis of paraffin embedded Human liver tissue. 1: Amyloid-β 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 98585-a Host Rabbit Application IHC-P Clonality Polyclonal Model Number STJ98585 Clone ID NA Antibody Name Anti-Amyloid-β 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
  • 2. Figure: Immunohistochemical analysis of paraffin embedded Rat lung tissue. 1: Amyloid-β 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 98585-b Host Rabbit Application IHC-P Clonality Polyclonal Model Number STJ98585 Clone ID NA Antibody Name Anti-Amyloid-β 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). 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
  • 3. Figure: Immunohistochemical analysis of paraffin embedded Rat kidney tissue. 1: Amyloid-β 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 98585-c Host Rabbit Application IHC-P Clonality Polyclonal Model Number STJ98585 Clone ID NA Antibody Name Anti-Amyloid-β 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). 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
  • 4. Figure: Immunohistochemical analysis of paraffin embedded Rat spleen tissue. 1: Amyloid-β 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 98585-d Host Rabbit Application IHC-P Clonality Polyclonal Model Number STJ98585 Clone ID NA Antibody Name Anti-Amyloid-β 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). 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
  • 5. Figure: Immunohistochemical analysis of paraffin embedded Mouse heart tissue. 1: Amyloid-β 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 98585-e Host Rabbit Application IHC-P Clonality Polyclonal Model Number STJ98585 Clone ID NA Antibody Name Anti-Amyloid-β antibody Testing Species MOUSE Testing Tissue HEART ANTIBODY VALIDATION REPORT c. (A small amount of distilled water was added into the incubation box to prevent evaporation of antibody). 45. 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. 46. 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. 47. 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. 48. 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. 49. Visualization a. Results were validated with microscope, and the slides were scanned. Paraffin-Embedded Immunohistochemistry Protocol 50. 51. 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. 52. 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. 53. 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. 54. 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. 55. 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 Mouse liver tissue. 1: Amyloid-β 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 98585-f Host Rabbit Application IHC-P Clonality Polyclonal Model Number STJ98585 Clone ID NA Antibody Name Anti-Amyloid-β antibody Testing Species MOUSE Testing Tissue LIVER ANTIBODY VALIDATION REPORT b. (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 kidney tissue. 1: Amyloid-β 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 98585-g Host Rabbit Application IHC-P Clonality Polyclonal Model Number STJ98585 Clone ID NA Antibody Name Anti-Amyloid-β 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). 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