This antibody validation report describes the validation of a polyclonal rabbit anti-HSP A5 antibody for use in immunofluorescence analysis of rat and mouse tissue samples. The report provides details of the immunofluorescence protocol used, including tissue processing, antigen retrieval, antibody incubations, counterstaining, mounting and visualization. Images show immunofluorescence staining of rat lung, spleen, and mouse lung tissues with the anti-HSP A5 antibody and DAPI nuclear counterstain.

1. ANTIBODY VALIDATION REPORT
Report Number 93610-a
Application Immunofluorescence
Model Number STJ93610
Antibody Name Anti-HSP A5 antibody
Host Rabbit
Clonality Polyclonal
Clone ID NA
Species RAT Tissue LUNG
Image
Description
Immunofluorescence analysis of Rat
lung tissue. 1: HSP A5 Polyclonal
Antibody(red) was diluted at 1:200 (4
degree Celsius,overnight). 2: Cy3 labled
Secondary antibody was diluted at
1:300 (room temperature, 50min).3:
Picture B: DAPI(blue) 10min. Picture
A:Target. Picture B: DAPI. Picture C:
merge of A+B.
Primary Antibody Incubation
After blocking solution was removed a 1:200 primary antibody/PBS
solution was added on the slide, and incubated overnight at 4°C (a
small amount of distilled water was added into the incubation box to
prevent evaporation of antibody).
Secondary Antibody Incubation
slides were washed with PBS on a shaker for 5min, and repeated 3
times. Shortly after the slides were dried and corresponding
secondary antibody solution was added (HRP labelled), covering the
tissues, and incubated at room temperature for 50min.
DAPI Counter-Staining
slides were washed with PBS on a shaker for 5min, repeated 3 times
and then dried. DAPI staining solution was added inside the PAP
circles and incubated for 10 min at room temperature without light
exposure.
Mounting
Slides were washed with PBS on a shaker for 5min, and repeated 3
times. Shortly after slides were dried, anti-quench mountings were
used to mount slides.
Visualization
The slides were observed and placed under a NIKON inverted
fluorescence microscope (Ultra violet excitation 330-380nm,
emission 420nm; FITC green excitation 465-495nm, emission 515-
555 nm; CY3 red excitation 510-560nm, emission 590nm)
Immunofluorescence Protocol
Tissue Processing
Slides were incubated sequentially into: Xylene - 15min, Anhydrous
ethanol – 15 min, Anhydrous ethanol – 5 min, 85% alcohol – 5 min,
75% alcohol – 5 min & washed with distilled water – 5 min.
Antigen Retrieval
Tissue slides were incubated with citric acid (PH6.0) antigen
retrieval buffer, and microwaved for antigen retrieval (heated until
boiled and then stop heating) for 8min. Slides were then heated with
medium power for 7min. During this process slides are kept from
drying out. After cooling down at room temperature, slides were
washed with PBS on a shaker for 5min, and repeated 3 times.
Anti-Quench
shortly after slides were dried, a PAP pen was used to draw circles
around the tissues (to prevent draining of the antibody). Inside the
circles, anti-quench mountings were added and incubated for 5 min,
and then flushed with water for 10min.
BSA Blocking
Inside the circles, BSA was used to cover the tissue evenly, blocking
for 30min.

2. ANTIBODY VALIDATION REPORT
Report Number 93610-b
Application Immunofluorescence
Model Number STJ93610
Antibody Name Anti-HSP A5 antibody
Host Rabbit
Clonality Polyclonal
Clone ID NA
Species RAT Tissue LUNG
Image
Description
Immunofluorescence analysis of Rat
lung tissue. 1: HSP A5 Polyclonal
Antibody(red) was diluted at 1:200 (4
degree Celsius,overnight). 2: Cy3 labled
Secondary antibody was diluted at
1:300 (room temperature, 50min).3:
Picture B: DAPI(blue) 10min. Picture
A:Target. Picture B: DAPI. Picture C:
merge of A+B.
Primary Antibody Incubation
After blocking solution was removed a 1:200 primary antibody/PBS
solution was added on the slide, and incubated overnight at 4°C (a
small amount of distilled water was added into the incubation box to
prevent evaporation of antibody).
Secondary Antibody Incubation
slides were washed with PBS on a shaker for 5min, and repeated 3
times. Shortly after the slides were dried and corresponding
secondary antibody solution was added (HRP labelled), covering the
tissues, and incubated at room temperature for 50min.
DAPI Counter-Staining
slides were washed with PBS on a shaker for 5min, repeated 3 times
and then dried. DAPI staining solution was added inside the PAP
circles and incubated for 10 min at room temperature without light
exposure.
Mounting
Slides were washed with PBS on a shaker for 5min, and repeated 3
times. Shortly after slides were dried, anti-quench mountings were
used to mount slides.
Visualization
The slides were observed and placed under a NIKON inverted
fluorescence microscope (Ultra violet excitation 330-380nm,
emission 420nm; FITC green excitation 465-495nm, emission 515-
555 nm; CY3 red excitation 510-560nm, emission 590nm)
Immunofluorescence Protocol
Tissue Processing
Slides were incubated sequentially into: Xylene - 15min, Anhydrous
ethanol – 15 min, Anhydrous ethanol – 5 min, 85% alcohol – 5 min,
75% alcohol – 5 min & washed with distilled water – 5 min.
Antigen Retrieval
Tissue slides were incubated with citric acid (PH6.0) antigen
retrieval buffer, and microwaved for antigen retrieval (heated until
boiled and then stop heating) for 8min. Slides were then heated with
medium power for 7min. During this process slides are kept from
drying out. After cooling down at room temperature, slides were
washed with PBS on a shaker for 5min, and repeated 3 times.
Anti-Quench
shortly after slides were dried, a PAP pen was used to draw circles
around the tissues (to prevent draining of the antibody). Inside the
circles, anti-quench mountings were added and incubated for 5 min,
and then flushed with water for 10min.
BSA Blocking
Inside the circles, BSA was used to cover the tissue evenly, blocking
for 30min.

3. ANTIBODY VALIDATION REPORT
Report Number 93610-c
Application Immunofluorescence
Model Number STJ93610
Antibody Name Anti-HSP A5 antibody
Host Rabbit
Clonality Polyclonal
Clone ID NA
Species RAT Tissue SPLEEN
Image
Description
Immunofluorescence analysis of Rat
spleen tissue. 1: HSP A5 Polyclonal
Antibody(red) was diluted at 1:200 (4
degree Celsius,overnight). 2: Cy3 labled
Secondary antibody was diluted at
1:300 (room temperature, 50min).3:
Picture B: DAPI(blue) 10min. Picture
A:Target. Picture B: DAPI. Picture C:
merge of A+B.
Primary Antibody Incubation
After blocking solution was removed a 1:200 primary antibody/PBS
solution was added on the slide, and incubated overnight at 4°C (a
small amount of distilled water was added into the incubation box to
prevent evaporation of antibody).
Secondary Antibody Incubation
slides were washed with PBS on a shaker for 5min, and repeated 3
times. Shortly after the slides were dried and corresponding
secondary antibody solution was added (HRP labelled), covering the
tissues, and incubated at room temperature for 50min.
DAPI Counter-Staining
slides were washed with PBS on a shaker for 5min, repeated 3 times
and then dried. DAPI staining solution was added inside the PAP
circles and incubated for 10 min at room temperature without light
exposure.
Mounting
Slides were washed with PBS on a shaker for 5min, and repeated 3
times. Shortly after slides were dried, anti-quench mountings were
used to mount slides.
Visualization
The slides were observed and placed under a NIKON inverted
fluorescence microscope (Ultra violet excitation 330-380nm,
emission 420nm; FITC green excitation 465-495nm, emission 515-
555 nm; CY3 red excitation 510-560nm, emission 590nm)
Immunofluorescence Protocol
Tissue Processing
Slides were incubated sequentially into: Xylene - 15min, Anhydrous
ethanol – 15 min, Anhydrous ethanol – 5 min, 85% alcohol – 5 min,
75% alcohol – 5 min & washed with distilled water – 5 min.
Antigen Retrieval
Tissue slides were incubated with citric acid (PH6.0) antigen
retrieval buffer, and microwaved for antigen retrieval (heated until
boiled and then stop heating) for 8min. Slides were then heated with
medium power for 7min. During this process slides are kept from
drying out. After cooling down at room temperature, slides were
washed with PBS on a shaker for 5min, and repeated 3 times.
Anti-Quench
shortly after slides were dried, a PAP pen was used to draw circles
around the tissues (to prevent draining of the antibody). Inside the
circles, anti-quench mountings were added and incubated for 5 min,
and then flushed with water for 10min.
BSA Blocking
Inside the circles, BSA was used to cover the tissue evenly, blocking
for 30min.

4. ANTIBODY VALIDATION REPORT
Report Number 93610-d
Application Immunofluorescence
Model Number STJ93610
Antibody Name Anti-HSP A5 antibody
Host Rabbit
Clonality Polyclonal
Clone ID NA
Species RAT Tissue SPLEEN
Image
Description
Immunofluorescence analysis of Rat
spleen tissue. 1: HSP A5 Polyclonal
Antibody(red) was diluted at 1:200 (4
degree Celsius,overnight). 2: Cy3 labled
Secondary antibody was diluted at
1:300 (room temperature, 50min).3:
Picture B: DAPI(blue) 10min. Picture
A:Target. Picture B: DAPI. Picture C:
merge of A+B.
Primary Antibody Incubation
After blocking solution was removed a 1:200 primary antibody/PBS
solution was added on the slide, and incubated overnight at 4°C (a
small amount of distilled water was added into the incubation box to
prevent evaporation of antibody).
Secondary Antibody Incubation
slides were washed with PBS on a shaker for 5min, and repeated 3
times. Shortly after the slides were dried and corresponding
secondary antibody solution was added (HRP labelled), covering the
tissues, and incubated at room temperature for 50min.
DAPI Counter-Staining
slides were washed with PBS on a shaker for 5min, repeated 3 times
and then dried. DAPI staining solution was added inside the PAP
circles and incubated for 10 min at room temperature without light
exposure.
Mounting
Slides were washed with PBS on a shaker for 5min, and repeated 3
times. Shortly after slides were dried, anti-quench mountings were
used to mount slides.
Visualization
The slides were observed and placed under a NIKON inverted
fluorescence microscope (Ultra violet excitation 330-380nm,
emission 420nm; FITC green excitation 465-495nm, emission 515-
555 nm; CY3 red excitation 510-560nm, emission 590nm)
Immunofluorescence Protocol
Tissue Processing
Slides were incubated sequentially into: Xylene - 15min, Anhydrous
ethanol – 15 min, Anhydrous ethanol – 5 min, 85% alcohol – 5 min,
75% alcohol – 5 min & washed with distilled water – 5 min.
Antigen Retrieval
Tissue slides were incubated with citric acid (PH6.0) antigen
retrieval buffer, and microwaved for antigen retrieval (heated until
boiled and then stop heating) for 8min. Slides were then heated with
medium power for 7min. During this process slides are kept from
drying out. After cooling down at room temperature, slides were
washed with PBS on a shaker for 5min, and repeated 3 times.
Anti-Quench
shortly after slides were dried, a PAP pen was used to draw circles
around the tissues (to prevent draining of the antibody). Inside the
circles, anti-quench mountings were added and incubated for 5 min,
and then flushed with water for 10min.
BSA Blocking
Inside the circles, BSA was used to cover the tissue evenly, blocking
for 30min.

5. ANTIBODY VALIDATION REPORT
Report Number 93610-e
Application Immunofluorescence
Model Number STJ93610
Antibody Name Anti-HSP A5 antibody
Host Rabbit
Clonality Polyclonal
Clone ID NA
Species MOUSE Tissue LUNG
Image
Description
Immunofluorescence analysis of Mouse
lung tissue. 1: HSP A5 Polyclonal
Antibody(red) was diluted at 1:200 (4
degree Celsius,overnight). 2: Cy3 labled
Secondary antibody was diluted at
1:300 (room temperature, 50min).3:
Picture B: DAPI(blue) 10min. Picture
A:Target. Picture B: DAPI. Picture C:
merge of A+B.
Primary Antibody Incubation
After blocking solution was removed a 1:200 primary antibody/PBS
solution was added on the slide, and incubated overnight at 4°C (a
small amount of distilled water was added into the incubation box to
prevent evaporation of antibody).
Secondary Antibody Incubation
slides were washed with PBS on a shaker for 5min, and repeated 3
times. Shortly after the slides were dried and corresponding
secondary antibody solution was added (HRP labelled), covering the
tissues, and incubated at room temperature for 50min.
DAPI Counter-Staining
slides were washed with PBS on a shaker for 5min, repeated 3 times
and then dried. DAPI staining solution was added inside the PAP
circles and incubated for 10 min at room temperature without light
exposure.
Mounting
Slides were washed with PBS on a shaker for 5min, and repeated 3
times. Shortly after slides were dried, anti-quench mountings were
used to mount slides.
Visualization
The slides were observed and placed under a NIKON inverted
fluorescence microscope (Ultra violet excitation 330-380nm,
emission 420nm; FITC green excitation 465-495nm, emission 515-
555 nm; CY3 red excitation 510-560nm, emission 590nm)
Immunofluorescence Protocol
Tissue Processing
Slides were incubated sequentially into: Xylene - 15min, Anhydrous
ethanol – 15 min, Anhydrous ethanol – 5 min, 85% alcohol – 5 min,
75% alcohol – 5 min & washed with distilled water – 5 min.
Antigen Retrieval
Tissue slides were incubated with citric acid (PH6.0) antigen
retrieval buffer, and microwaved for antigen retrieval (heated until
boiled and then stop heating) for 8min. Slides were then heated with
medium power for 7min. During this process slides are kept from
drying out. After cooling down at room temperature, slides were
washed with PBS on a shaker for 5min, and repeated 3 times.
Anti-Quench
shortly after slides were dried, a PAP pen was used to draw circles
around the tissues (to prevent draining of the antibody). Inside the
circles, anti-quench mountings were added and incubated for 5 min,
and then flushed with water for 10min.
BSA Blocking
Inside the circles, BSA was used to cover the tissue evenly, blocking
for 30min.

6. ANTIBODY VALIDATION REPORT
Report Number 93610-f
Application Immunofluorescence
Model Number STJ93610
Antibody Name Anti-HSP A5 antibody
Host Rabbit
Clonality Polyclonal
Clone ID NA
Species MOUSE Tissue LUNG
Image
Description
Immunofluorescence analysis of Mouse
lung tissue. 1: HSP A5 Polyclonal
Antibody(red) was diluted at 1:200 (4
degree Celsius,overnight). 2: Cy3 labled
Secondary antibody was diluted at
1:300 (room temperature, 50min).3:
Picture B: DAPI(blue) 10min. Picture
A:Target. Picture B: DAPI. Picture C:
merge of A+B.
Primary Antibody Incubation
After blocking solution was removed a 1:200 primary antibody/PBS
solution was added on the slide, and incubated overnight at 4°C (a
small amount of distilled water was added into the incubation box to
prevent evaporation of antibody).
Secondary Antibody Incubation
slides were washed with PBS on a shaker for 5min, and repeated 3
times. Shortly after the slides were dried and corresponding
secondary antibody solution was added (HRP labelled), covering the
tissues, and incubated at room temperature for 50min.
DAPI Counter-Staining
slides were washed with PBS on a shaker for 5min, repeated 3 times
and then dried. DAPI staining solution was added inside the PAP
circles and incubated for 10 min at room temperature without light
exposure.
Mounting
Slides were washed with PBS on a shaker for 5min, and repeated 3
times. Shortly after slides were dried, anti-quench mountings were
used to mount slides.
Visualization
The slides were observed and placed under a NIKON inverted
fluorescence microscope (Ultra violet excitation 330-380nm,
emission 420nm; FITC green excitation 465-495nm, emission 515-
555 nm; CY3 red excitation 510-560nm, emission 590nm)
Immunofluorescence Protocol
Tissue Processing
Slides were incubated sequentially into: Xylene - 15min, Anhydrous
ethanol – 15 min, Anhydrous ethanol – 5 min, 85% alcohol – 5 min,
75% alcohol – 5 min & washed with distilled water – 5 min.
Antigen Retrieval
Tissue slides were incubated with citric acid (PH6.0) antigen
retrieval buffer, and microwaved for antigen retrieval (heated until
boiled and then stop heating) for 8min. Slides were then heated with
medium power for 7min. During this process slides are kept from
drying out. After cooling down at room temperature, slides were
washed with PBS on a shaker for 5min, and repeated 3 times.
Anti-Quench
shortly after slides were dried, a PAP pen was used to draw circles
around the tissues (to prevent draining of the antibody). Inside the
circles, anti-quench mountings were added and incubated for 5 min,
and then flushed with water for 10min.
BSA Blocking
Inside the circles, BSA was used to cover the tissue evenly, blocking
for 30min.