Transcriptional activator which triggers apoptosis in the absence of survival factors, including neuronal cell death upon oxidative stress. Recognizes and binds to the DNA sequence 5'-[AG]TAAA[TC]A-3'. Participates in post-transcriptional regulation of MYC: following phosphorylation by MAPKAPK5, promotes induction of miR-34b and miR-34c expression, 2 post-transcriptional regulators of MYC that bind to the 3'UTR of MYC transcript and prevent its translation Anti-Phospho-FoxO3A (S253)-http://www.stjohnslabs.com/phospho-foxo3a-s253-antibody Join our Antibody Validation Project - http://www.stjohnslabs.com/services/antibody-validation

1. Figure:
Immunohistochemical
analysis of paraffin
embedded Human colon
tissue. 1: FoxO3A
(phospho Ser253)
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 90275-a Host Rabbit
Application IHC-P Clonality Polyclonal
Model Number STJ90275 Clone ID NA
Antibody Name Anti-Phospho-FoxO3A (S253) antibody
Testing Species HUMAN Testing Tissue COLON
ANTIBODY VALIDATION REPORT
a. (A small amount of distilled water was added into the incubation
box to prevent evaporation of antibody).
51. Secondary antibody incubation
a. Slides were washed 3 times, with PBS on a shaker for 5min.
Shortly after the slides were dried the corresponding secondary
antibody solution was added (HRP labelled), covering the
tissues, and incubated at room temperature for 30min.
b.
52. DAB staining
a. Slides were washed 3 times, with PBS on a shaker for 5min.
b. Shortly after, the slides were dried and fresh DAB staining buffer
was added inside the circles. The staining time was adjusted
under a microscope. Yellow-brown colour represented a positive
result. Slides were washed with water to stop the staining.
c.
53. Haematoxylin staining
a. Haematoxylin was used to counter-staining for 1min, and then
the slides were washed with water. 1% Hydrochloric acid and
alcohol was added for several seconds and then washed with
water. Ammonia was used to reveal blue colour, and then
flushed with water.
b.
54. Desolation and Clearing
i. Slides were incubated sequentially into: 75% alcohol 5min, 85%
alcohol 5min, Anhydrous ethanol - 5min, Anhydrous ethanol -
5min & Xylene - 5min. Shortly after slides were dried and neutral
gum was used to seal the slides.
ii.
55. Visualization
a. Results were validated with microscope, and the slides were
scanned.
Paraffin-Embedded
Immunohistochemistry Protocol
45.
46. Tissue processing
a. Slides were incubated sequentially into Xylene; 15min –
Xylene, 15min - Anhydrous ethanol, 5min - Anhydrous
ethanol, 5min - 85% alcohol, 5min - 75% alcohol & 5min –
wash in distilled water.
b.
47. Antigen retrieval
a. Tissue slides were incubated with citric acid (PH6.0) antigen
retrieval buffer and microwaved for antigen retrieval (heated
until boiled and then stopped heating) for 8min. Slides were
then heated with medium power for 7min. During this
process slides were kept from drying out. After cooling down
at room temperature, slides were washed with PBS on
shaker for 5min, repeated for 3 times.
b.
48. Inhibition of endogenous peroxidase
a. Slides were placed in 3% Hydrogen peroxide solution, and
incubated for 10 min at room temperature without light
exposure. Slides were then washed 3 times with PBS on a
shaker for 5mins.
b.
49. BSA Blocking
a. Shortly after slides were dried, a PAP pen was used to draw
circles around the tissue sections (and to prevent draining of
the antibody solution). Inside the circles, BSA was used to
cover the tissue evenly, blocking for 30min.
b.
50. Primary antibody incubation
After blocking solution was removed a 1:200 solution of
primary antibody/PBS was added on the slide, and incubated
overnight at 4°C.
St John's Laboratory Ltd.
www.stjohnslabs.com

2. Figure:
Immunohistochemical
analysis of paraffin
embedded Rat testis
tissue. 1: FoxO3A
(phospho Ser253)
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 90275-b Host Rabbit
Application IHC-P Clonality Polyclonal
Model Number STJ90275 Clone ID NA
Antibody Name Anti-Phospho-FoxO3A (S253) antibody
Testing Species RAT Testing Tissue TESTIS
ANTIBODY VALIDATION REPORT
a. (A small amount of distilled water was added into the incubation
box to prevent evaporation of antibody).
40. Secondary antibody incubation
a. Slides were washed 3 times, with PBS on a shaker for 5min.
Shortly after the slides were dried the corresponding secondary
antibody solution was added (HRP labelled), covering the
tissues, and incubated at room temperature for 30min.
b.
41. DAB staining
a. Slides were washed 3 times, with PBS on a shaker for 5min.
b. Shortly after, the slides were dried and fresh DAB staining buffer
was added inside the circles. The staining time was adjusted
under a microscope. Yellow-brown colour represented a positive
result. Slides were washed with water to stop the staining.
c.
42. Haematoxylin staining
a. Haematoxylin was used to counter-staining for 1min, and then
the slides were washed with water. 1% Hydrochloric acid and
alcohol was added for several seconds and then washed with
water. Ammonia was used to reveal blue colour, and then
flushed with water.
b.
43. Desolation and Clearing
i. Slides were incubated sequentially into: 75% alcohol 5min, 85%
alcohol 5min, Anhydrous ethanol - 5min, Anhydrous ethanol -
5min & Xylene - 5min. Shortly after slides were dried and neutral
gum was used to seal the slides.
ii.
44. Visualization
a. Results were validated with microscope, and the slides were
scanned.
Paraffin-Embedded
Immunohistochemistry Protocol
34.
35. Tissue processing
a. Slides were incubated sequentially into Xylene; 15min –
Xylene, 15min - Anhydrous ethanol, 5min - Anhydrous
ethanol, 5min - 85% alcohol, 5min - 75% alcohol & 5min –
wash in distilled water.
b.
36. Antigen retrieval
a. Tissue slides were incubated with citric acid (PH6.0) antigen
retrieval buffer and microwaved for antigen retrieval (heated
until boiled and then stopped heating) for 8min. Slides were
then heated with medium power for 7min. During this
process slides were kept from drying out. After cooling down
at room temperature, slides were washed with PBS on
shaker for 5min, repeated for 3 times.
b.
37. Inhibition of endogenous peroxidase
a. Slides were placed in 3% Hydrogen peroxide solution, and
incubated for 10 min at room temperature without light
exposure. Slides were then washed 3 times with PBS on a
shaker for 5mins.
b.
38. BSA Blocking
a. Shortly after slides were dried, a PAP pen was used to draw
circles around the tissue sections (and to prevent draining of
the antibody solution). Inside the circles, BSA was used to
cover the tissue evenly, blocking for 30min.
b.
39. Primary antibody incubation
After blocking solution was removed a 1:200 solution of
primary antibody/PBS was added on the slide, and incubated
overnight at 4°C.
St John's Laboratory Ltd.
www.stjohnslabs.com

3. Figure:
Immunohistochemical
analysis of paraffin
embedded Rat lung
tissue. 1: FoxO3A
(phospho Ser253)
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 90275-c Host Rabbit
Application IHC-P Clonality Polyclonal
Model Number STJ90275 Clone ID NA
Antibody Name Anti-Phospho-FoxO3A (S253) 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

4. Figure:
Immunohistochemical
analysis of paraffin
embedded Rat spleen
tissue. 1: FoxO3A
(phospho Ser253)
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 90275-d Host Rabbit
Application IHC-P Clonality Polyclonal
Model Number STJ90275 Clone ID NA
Antibody Name Anti-Phospho-FoxO3A (S253) antibody
Testing Species RAT Testing Tissue SPLEEN
ANTIBODY VALIDATION REPORT
a. (A small amount of distilled water was added into the incubation
box to prevent evaporation of antibody).
18. Secondary antibody incubation
a. Slides were washed 3 times, with PBS on a shaker for 5min.
Shortly after the slides were dried the corresponding secondary
antibody solution was added (HRP labelled), covering the
tissues, and incubated at room temperature for 30min.
b.
19. DAB staining
a. Slides were washed 3 times, with PBS on a shaker for 5min.
b. Shortly after, the slides were dried and fresh DAB staining buffer
was added inside the circles. The staining time was adjusted
under a microscope. Yellow-brown colour represented a positive
result. Slides were washed with water to stop the staining.
c.
20. Haematoxylin staining
a. Haematoxylin was used to counter-staining for 1min, and then
the slides were washed with water. 1% Hydrochloric acid and
alcohol was added for several seconds and then washed with
water. Ammonia was used to reveal blue colour, and then
flushed with water.
b.
21. Desolation and Clearing
i. Slides were incubated sequentially into: 75% alcohol 5min, 85%
alcohol 5min, Anhydrous ethanol - 5min, Anhydrous ethanol -
5min & Xylene - 5min. Shortly after slides were dried and neutral
gum was used to seal the slides.
ii.
22. Visualization
a. Results were validated with microscope, and the slides were
scanned.
Paraffin-Embedded
Immunohistochemistry Protocol
12.
13. Tissue processing
a. Slides were incubated sequentially into Xylene; 15min –
Xylene, 15min - Anhydrous ethanol, 5min - Anhydrous
ethanol, 5min - 85% alcohol, 5min - 75% alcohol & 5min –
wash in distilled water.
b.
14. Antigen retrieval
a. Tissue slides were incubated with citric acid (PH6.0) antigen
retrieval buffer and microwaved for antigen retrieval (heated
until boiled and then stopped heating) for 8min. Slides were
then heated with medium power for 7min. During this
process slides were kept from drying out. After cooling down
at room temperature, slides were washed with PBS on
shaker for 5min, repeated for 3 times.
b.
15. Inhibition of endogenous peroxidase
a. Slides were placed in 3% Hydrogen peroxide solution, and
incubated for 10 min at room temperature without light
exposure. Slides were then washed 3 times with PBS on a
shaker for 5mins.
b.
16. BSA Blocking
a. Shortly after slides were dried, a PAP pen was used to draw
circles around the tissue sections (and to prevent draining of
the antibody solution). Inside the circles, BSA was used to
cover the tissue evenly, blocking for 30min.
b.
17. Primary antibody incubation
After blocking solution was removed a 1:200 solution of
primary antibody/PBS was added on the slide, and incubated
overnight at 4°C.
St John's Laboratory Ltd.
www.stjohnslabs.com

5. Figure:
Immunohistochemical
analysis of paraffin
embedded Mouse testis
tissue. 1: FoxO3A
(phospho Ser253)
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 90275-e Host Rabbit
Application IHC-P Clonality Polyclonal
Model Number STJ90275 Clone ID NA
Antibody Name Anti-Phospho-FoxO3A (S253) antibody
Testing Species MOUSE Testing Tissue TESTIS
ANTIBODY VALIDATION REPORT
a. (A small amount of distilled water was added into the incubation
box to prevent evaporation of antibody).
7. Secondary antibody incubation
a. Slides were washed 3 times, with PBS on a shaker for 5min.
Shortly after the slides were dried the corresponding secondary
antibody solution was added (HRP labelled), covering the
tissues, and incubated at room temperature for 30min.
b.
8. DAB staining
a. Slides were washed 3 times, with PBS on a shaker for 5min.
b. Shortly after, the slides were dried and fresh DAB staining buffer
was added inside the circles. The staining time was adjusted
under a microscope. Yellow-brown colour represented a positive
result. Slides were washed with water to stop the staining.
c.
9. Haematoxylin staining
a. Haematoxylin was used to counter-staining for 1min, and then
the slides were washed with water. 1% Hydrochloric acid and
alcohol was added for several seconds and then washed with
water. Ammonia was used to reveal blue colour, and then
flushed with water.
b.
10. Desolation and Clearing
i. Slides were incubated sequentially into: 75% alcohol 5min, 85%
alcohol 5min, Anhydrous ethanol - 5min, Anhydrous ethanol -
5min & Xylene - 5min. Shortly after slides were dried and neutral
gum was used to seal the slides.
ii.
11. Visualization
a. Results were validated with microscope, and the slides were
scanned.
Paraffin-Embedded
Immunohistochemistry Protocol
1.
2. Tissue processing
a. Slides were incubated sequentially into Xylene; 15min –
Xylene, 15min - Anhydrous ethanol, 5min - Anhydrous
ethanol, 5min - 85% alcohol, 5min - 75% alcohol & 5min –
wash in distilled water.
b.
3. Antigen retrieval
a. Tissue slides were incubated with citric acid (PH6.0) antigen
retrieval buffer and microwaved for antigen retrieval (heated
until boiled and then stopped heating) for 8min. Slides were
then heated with medium power for 7min. During this
process slides were kept from drying out. After cooling down
at room temperature, slides were washed with PBS on
shaker for 5min, repeated for 3 times.
b.
4. Inhibition of endogenous peroxidase
a. Slides were placed in 3% Hydrogen peroxide solution, and
incubated for 10 min at room temperature without light
exposure. Slides were then washed 3 times with PBS on a
shaker for 5mins.
b.
5. BSA Blocking
a. Shortly after slides were dried, a PAP pen was used to draw
circles around the tissue sections (and to prevent draining of
the antibody solution). Inside the circles, BSA was used to
cover the tissue evenly, blocking for 30min.
b.
6. Primary antibody incubation
After blocking solution was removed a 1:200 solution of
primary antibody/PBS was added on the slide, and incubated
overnight at 4°C.
St John's Laboratory Ltd.
www.stjohnslabs.com

6. Figure:
Immunohistochemical
analysis of paraffin
embedded Mouse liver
tissue. 1: FoxO3A
(phospho Ser253)
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 90275-f Host Rabbit
Application IHC-P Clonality Polyclonal
Model Number STJ90275 Clone ID NA
Antibody Name Anti-Phospho-FoxO3A (S253) antibody
Testing Species MOUSE Testing Tissue LIVER
ANTIBODY VALIDATION REPORT
c. (A small amount of distilled water was added into the incubation
box to prevent evaporation of antibody).
56. Secondary antibody incubation
a. Slides were washed 3 times, with PBS on a shaker for 5min.
Shortly after the slides were dried the corresponding secondary
antibody solution was added (HRP labelled), covering the
tissues, and incubated at room temperature for 30min.
b.
57. DAB staining
a. Slides were washed 3 times, with PBS on a shaker for 5min.
b. Shortly after, the slides were dried and fresh DAB staining buffer
was added inside the circles. The staining time was adjusted
under a microscope. Yellow-brown colour represented a positive
result. Slides were washed with water to stop the staining.
c.
58. Haematoxylin staining
a. Haematoxylin was used to counter-staining for 1min, and then
the slides were washed with water. 1% Hydrochloric acid and
alcohol was added for several seconds and then washed with
water. Ammonia was used to reveal blue colour, and then
flushed with water.
b.
59. Desolation and Clearing
i. Slides were incubated sequentially into: 75% alcohol 5min, 85%
alcohol 5min, Anhydrous ethanol - 5min, Anhydrous ethanol -
5min & Xylene - 5min. Shortly after slides were dried and neutral
gum was used to seal the slides.
ii.
60. Visualization
a. Results were validated with microscope, and the slides were
scanned.
Paraffin-Embedded
Immunohistochemistry Protocol
61.
62. Tissue processing
a. Slides were incubated sequentially into Xylene; 15min –
Xylene, 15min - Anhydrous ethanol, 5min - Anhydrous
ethanol, 5min - 85% alcohol, 5min - 75% alcohol & 5min –
wash in distilled water.
b.
63. Antigen retrieval
a. Tissue slides were incubated with citric acid (PH6.0) antigen
retrieval buffer and microwaved for antigen retrieval (heated
until boiled and then stopped heating) for 8min. Slides were
then heated with medium power for 7min. During this
process slides were kept from drying out. After cooling down
at room temperature, slides were washed with PBS on
shaker for 5min, repeated for 3 times.
b.
64. Inhibition of endogenous peroxidase
a. Slides were placed in 3% Hydrogen peroxide solution, and
incubated for 10 min at room temperature without light
exposure. Slides were then washed 3 times with PBS on a
shaker for 5mins.
b.
65. BSA Blocking
a. Shortly after slides were dried, a PAP pen was used to draw
circles around the tissue sections (and to prevent draining of
the antibody solution). Inside the circles, BSA was used to
cover the tissue evenly, blocking for 30min.
b.
66. Primary antibody incubation
After blocking solution was removed a 1:200 solution of
primary antibody/PBS was added on the slide, and incubated
overnight at 4°C.
St John's Laboratory Ltd.
www.stjohnslabs.com

7. Figure:
Immunohistochemical
analysis of paraffin
embedded Mouse colon
tissue. 1: FoxO3A
(phospho Ser253)
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 90275-g Host Rabbit
Application IHC-P Clonality Polyclonal
Model Number STJ90275 Clone ID NA
Antibody Name Anti-Phospho-FoxO3A (S253) antibody
Testing Species MOUSE Testing Tissue COLON
ANTIBODY VALIDATION REPORT
b. (A small amount of distilled water was added into the incubation
box to prevent evaporation of antibody).
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