ANTIBODY VALIDATION REPORT

Report Number 97051-a

Application Immunofluorescence

Model Number STJ97051

Antibody Name Anti-α skeletal muscle actin antibody

Host Mouse

Clonality Monoclonal

Clone ID 4B11

Species HUMAN Tissue LIVER CANCER

Image Description

Immunofluorescence analysis of Human

liver cancer tissue. 1: α skeletal muscle

actin Monoclonal Antibody(4B11)(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.

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

ANTIBODY VALIDATION REPORT

Report Number 97051-b

Application Immunofluorescence

Model Number STJ97051

Antibody Name Anti-α skeletal muscle actin antibody

Host Mouse

Clonality Monoclonal

Clone ID 4B11

Species HUMAN Tissue LIVER CANCER

Image Description

Immunofluorescence analysis of Human

liver cancer tissue. 1: α skeletal muscle

actin Monoclonal Antibody(4B11)(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.

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

ANTIBODY VALIDATION REPORT

Report Number 97051-c

Application Immunofluorescence

Model Number STJ97051

Antibody Name Anti-α skeletal muscle actin antibody

Host Mouse

Clonality Monoclonal

Clone ID 4B11

Species MOUSE Tissue LIVER

Image Description

Immunofluorescence analysis of Mouse

liver tissue. 1: α skeletal muscle actin

Monoclonal Antibody(4B11)(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.

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

ANTIBODY VALIDATION REPORT

Report Number 97051-d

Application Immunofluorescence

Model Number STJ97051

Antibody Name Anti-α skeletal muscle actin antibody

Host Mouse

Clonality Monoclonal

Clone ID 4B11

Species MOUSE Tissue LIVER

Image Description

Immunofluorescence analysis of Mouse

liver tissue. 1: α skeletal muscle actin

Monoclonal Antibody(4B11)(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.

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

ANTIBODY VALIDATION REPORT

Report Number 97051-e

Application Immunofluorescence

Model Number STJ97051

Antibody Name Anti-α skeletal muscle actin antibody

Host Mouse

Clonality Monoclonal

Clone ID 4B11

Species RAT Tissue LIVER

Image Description

Immunofluorescence analysis of Rat

liver tissue. 1: α skeletal muscle actin

Monoclonal Antibody(4B11)(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.

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

ANTIBODY VALIDATION REPORT

Report Number 97051-f

Application Immunofluorescence

Model Number STJ97051

Antibody Name Anti-α skeletal muscle actin antibody

Host Mouse

Clonality Monoclonal

Clone ID 4B11

Species RAT Tissue LIVER

Image Description

Immunofluorescence analysis of Rat

liver tissue. 1: α skeletal muscle actin

Monoclonal Antibody(4B11)(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.

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