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Figure: Immunohistochemical analysis of paraffin embedded Human uterus tissue. 1: IL-1β 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 96566-a Host Rabbit Application IHC-P Clonality Polyclonal Model Number STJ96566 Clone ID NA Antibody Name Anti-IL-1β antibody Testing Species HUMAN Testing Tissue UTERUS ANTIBODY VALIDATION REPORT (A small amount of distilled water was added into the incubation box to prevent evaporation of antibody). Secondary antibody incubation 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. DAB staining Slides were washed 3 times, with PBS on a shaker for 5min. 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. Haematoxylin staining 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. Desolation and Clearing 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. Visualization Results were validated with microscope, and the slides were scanned. Paraffin-Embedded Immunohistochemistry Protocol Tissue processing 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. Antigen retrieval 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. Inhibition of endogenous peroxidase 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. BSA Blocking 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. 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

Immunohistochemistry Antibody Validation Report for Anti-IL-1β Antibody (STJ96566)

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Text of Immunohistochemistry Antibody Validation Report for Anti-IL-1β Antibody (STJ96566)

  • Figure:

    Immunohistochemical

    analysis of paraffin

    embedded Human

    uterus tissue. 1: IL-1

    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 96566-a Host Rabbit

    Application IHC-P Clonality Polyclonal

    Model Number STJ96566 Clone ID NA

    Antibody Name Anti-IL-1 antibody

    Testing Species HUMAN Testing Tissue UTERUS

    ANTIBODY VALIDATION REPORT

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

    box to prevent evaporation of antibody).

    41. Secondary antibody incubation

    a. Slides were washed 3 times, with PBS on a shaker for 5min.

    Shortly after the slides were dried the corresponding secondary

    antibody solution was added (HRP labelled), covering the

    tissues, and incubated at room temperature for 30min.

    b.

    42. DAB staining

    a. Slides were washed 3 times, with PBS on a shaker for 5min.

    b. Shortly after, the slides were dried and fresh DAB staining buffer

    was added inside the circles. The staining time was adjusted

    under a microscope. Yellow-brown colour represented a positive

    result. Slides were washed with water to stop the staining.

    c.

    43. Haematoxylin staining

    a. Haematoxylin was used to counter-staining for 1min, and then

    the slides were washed with water. 1% Hydrochloric acid and

    alcohol was added for several seconds and then washed with

    water. Ammonia was used to reveal blue colour, and then

    flushed with water.

    b.

    44. Desolation and Clearing

    i. Slides were incubated sequentially into: 75% alcohol 5min, 85%

    alcohol 5min, Anhydrous ethanol - 5min, Anhydrous ethanol -

    5min & Xylene - 5min. Shortly after slides were dried and neutral

    gum was used to seal the slides.

    ii.

    45. Visualization

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

    scanned.

    Paraffin-Embedded

    Immunohistochemistry Protocol 35.

    36. Tissue processing

    a. Slides were incubated sequentially into Xylene; 15min

    Xylene, 15min - Anhydrous ethanol, 5min - Anhydrous

    ethanol, 5min - 85% alcohol, 5min - 75% alcohol & 5min

    wash in distilled water.

    b.

    37. Antigen retrieval

    a. Tissue slides were incubated with citric acid (PH6.0) antigen

    retrieval buffer and microwaved for antigen retrieval (heated

    until boiled and then stopped heating) for 8min. Slides were

    then heated with medium power for 7min. During this

    process slides were kept from drying out. After cooling down

    at room temperature, slides were washed with PBS on

    shaker for 5min, repeated for 3 times.

    b.

    38. Inhibition of endogenous peroxidase

    a. Slides were placed in 3% Hydrogen peroxide solution, and

    incubated for 10 min at room temperature without light

    exposure. Slides were then washed 3 times with PBS on a

    shaker for 5mins.

    b.

    39. BSA Blocking

    a. Shortly after slides were dried, a PAP pen was used to draw

    circles around the tissue sections (and to prevent draining of

    the antibody solution). Inside the circles, BSA was used to

    cover the tissue evenly, blocking for 30min.

    b.

    40. Primary antibody incubation

    After blocking solution was removed a 1:200 solution of

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

    overnight at 4C.

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

  • Figure:

    Immunohistochemical

    analysis of paraffin

    embedded Rat lung

    tissue. 1: IL-1

    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 96566-b Host Rabbit

    Application IHC-P Clonality Polyclonal

    Model Number STJ96566 Clone ID NA

    Antibody Name Anti-IL-1 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).

    30. Secondary antibody incubation

    a. Slides were washed 3 times, with PBS on a shaker for 5min.

    Shortly after the slides were dried the corresponding secondary

    antibody solution was added (HRP labelled), covering the

    tissues, and incubated at room temperature for 30min.

    b.

    31. DAB staining

    a. Slides were washed 3 times, with PBS on a shaker for 5min.

    b. Shortly after, the slides were dried and fresh DAB staining buffer

    was added inside the circles. The staining time was adjusted

    under a microscope. Yellow-brown colour represented a positive

    result. Slides were washed with water to stop the staining.

    c.

    32. Haematoxylin staining

    a. Haematoxylin was used to counter-staining for 1min, and then

    the slides were washed with water. 1% Hydrochloric acid and

    alcohol was added for several seconds and then washed with

    water. Ammonia was used to reveal blue colour, and then

    flushed with water.

    b.

    33. Desolation and Clearing

    i. Slides were incubated sequentially into: 75% alcohol 5min, 85%

    alcohol 5min, Anhydrous ethanol - 5min, Anhydrous ethanol -

    5min & Xylene - 5min. Shortly after slides were dried and neutral

    gum was used to seal the slides.

    ii.

    34. Visualization

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

    scanned.

    Paraffin-Embedded

    Immunohistochemistry Protocol 24.

    25. Tissue processing

    a. Slides were incubated sequentially into Xylene; 15min

    Xylene, 15min - Anhydrous ethanol, 5min - Anhydrous

    ethanol, 5min - 85% alcohol, 5min - 75% alcohol & 5min

    wash in distilled water.

    b.

    26. Antigen retrieval

    a. Tissue slides were incubated with citric acid (PH6.0) antigen

    retrieval buffer and microwaved for antigen retrieval (heated

    until boiled and then stopped heating) for 8min. Slides were

    then heated with medium power for 7min. During this

    process slides were kept from drying out. After cooling down

    at room temperature, slides were washed with PBS on

    shaker for 5min, repeated for 3 times.

    b.

    27. Inhibition of endogenous peroxidase

    a. Slides were placed in 3% Hydrogen peroxide solution, and

    incubated for 10 min at room temperature without light

    exposure. Slides were then washed 3 times with PBS on a

    shaker for 5mins.

    b.

    28. BSA Blocking

    a. Shortly after slides were dried, a PAP pen was used to draw

    circles around the tissue sections (and to prevent draining of

    the antibody solution). Inside the circles, BSA was used to

    cover the tissue evenly, blocking for 30min.

    b.

    29. Primary antibody incubation

    After blocking solution was removed a 1:200 solution of

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

    overnight at 4C.

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

  • Figure:

    Immunohistochemical

    analysis of paraffin

    embedded Mouse heart

    tissue. 1: IL-1

    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 96566-c Host Rabbit

    Application IHC-P Clonality Polyclonal

    Model Number STJ96566 Clone ID NA

    Antibody Name Anti-IL-1 antibody

    Testing Species MOUSE Testing Tissue HEART

    ANTIBODY VALIDATION REPORT

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

    box to prevent evaporation of antibody).

    19. Secondary antibody incubation

    a. Slides were washed 3 times, with PBS on a shaker for 5min.

    Shortly after the slides were dried the corresponding secondary

    antibody solution was added (HRP labelled), covering the

    tissues, and incubated at room temperature for 30min.

    b.

    20. DAB staining

    a. Slides were washed 3 times, with PBS on a shaker for 5min.

    b. Shortly after, the slides were dried and fresh DAB staining buffer

    was added inside the circles. The staining time was adjusted

    under a microscope. Yellow-brown colour represented a positive

    result. Slides were washed with water to stop the staining.

    c.

    21. Haematoxylin staining

    a. Haematoxylin was used to counter-staining for 1min, and then

    the slides were washed with water. 1% Hydrochloric acid and

    alcohol was added for several seconds and then washed with

    water. Ammonia was used to reveal blue colour, and then

    flushed with water.

    b.

    22. Desolation and Clearing

    i. Slides were incubated sequentially into: 75% alcohol 5min, 85%

    alcohol 5min, Anhydrous ethanol - 5min, Anhydrous ethanol -

    5min & Xylene - 5min. Shortly after slides were dried and neutral

    gum was used to seal the slides.

    ii.

    23. Visualization

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

    scanned.

    Paraffin-Embedded

    Immunohistochemistry Protocol 13.

    14. Tissue processing

    a. Slides were incubated sequentially into Xylene; 15min

    Xylene, 15min - Anhydrous ethanol, 5min - Anhydrous

    ethanol, 5min - 85% alcohol, 5min - 75% alcohol & 5min

    wash in distilled water.

    b.

    15. Antigen retrieval

    a. Tissue slides were incubated with citric acid (PH6.0) antigen

    retrieval buffer and microwaved for antigen retrieval (heated

    until boiled and then stopped heating) for 8min. Slides were

    then heated with medium power for 7min. During this

    process slides were kept from drying out. After cooling down

    at room temperature, slides were washed with PBS on

    shaker for 5min, repeated for 3 times.

    b.

    16. Inhibition of endogenous peroxidase

    a. Slides were placed in 3% Hydrogen peroxide solution, and

    incubated for 10 min at room temperature without light

    exposure. Slides were then washed 3 times with PBS on a

    shaker for 5mins.

    b.

    17. BSA Blocking

    a. Shortly after slides were dried, a PAP pen was used to draw

    circles around the tissue sections (and to prevent draining of

    the antibody solution). Inside the circles, BSA was used to

    cover the tissue evenly, blocking for 30min.

    b.

    18. Primary antibody incubation

    After blocking solution was removed a 1:200 solution of

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

    overnight at 4C.

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

  • Figure:

    Immunohistochemical

    analysis of paraffin

    embedded Mouse lung

    tissue. 1: IL-1

    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 96566-d Host Rabbit

    Application IHC-P Clonality Polyclonal

    Model Number STJ96566 Clone ID NA

    Antibody Name Anti-IL-1 antibody

    Testing Species MOUSE Testing Tissue LUNG

    ANTIBODY VALIDATION REPORT

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

    box to prevent evaporation of antibody).

    8. Secondary antibody incubation

    a. Slides were washed 3 times, with PBS on a shaker for 5min.

    Shortly after the slides were dried the corresponding secondary

    antibody solution was added (HRP labelled), covering the

    tissues, and incubated at room temperature for 30min.

    b.

    9. DAB staining

    a. Slides were washed 3 times, with PBS on a shaker for 5min.

    b. Shortly after, the slides were dried and fresh DAB staining buffer

    was added inside the circles. The staining time was adjusted

    under a microscope. Yellow-brown colour represented a positive

    result. Slides were washed with water to stop the staining.

    c.

    10. Haematoxylin staining

    a. Haematoxylin was used to counter-staining for 1min, and then

    the slides were washed with water. 1% Hydrochloric acid and

    alcohol was added for several seconds and then washed with

    water. Ammonia was used to reveal blue colour, and then

    flushed with water.

    b.

    11. Desolation and Clearing

    i. Slides were incubated sequentially into: 75% alcohol 5min, 85%

    alcohol 5min, Anhydrous ethanol - 5min, Anhydrous ethanol -

    5min & Xylene - 5min. Shortly after slides were dried and neutral

    gum was used to seal the slides.

    ii.

    12. Visualization

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

    scanned.

    Paraffin-Embedded

    Immunohistochemistry Protocol 2.

    3. Tissue processing

    a. Slides were incubated sequentially into Xylene; 15min

    Xylene, 15min - Anhydrous ethanol, 5min - Anhydrous

    ethanol, 5min - 85% alcohol, 5min - 75% alcohol & 5min

    wash in distilled water.

    b.

    4. Antigen retrieval

    a. Tissue slides were incubated with citric acid (PH6.0) antigen

    retrieval buffer and microwaved for antigen retrieval (heated

    until boiled and then stopped heating) for 8min. Slides were

    then heated with medium power for 7min. During this

    process slides were kept from drying out. After cooling down

    at room temperature, slides were washed with PBS on

    shaker for 5min, repeated for 3 times.

    b.

    5. Inhibition of endogenous peroxidase

    a. Slides were placed in 3% Hydrogen peroxide solution, and

    incubated for 10 min at room temperature without light

    exposure. Slides were then washed 3 times with PBS on a

    shaker for 5mins.

    b.

    6. BSA Blocking

    a. Shortly after slides were dried, a PAP pen was used to draw

    circles around the tissue sections (and to prevent draining of

    the antibody solution). Inside the circles, BSA was used to

    cover the tissue evenly, blocking for 30min.

    b.

    7. Primary antibody incubation

    After blocking solution was removed a 1:200 solution of

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

    overnight at 4C.

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