Immunohistochemistry Protocol for Keratin Antibodies

 

Introduction

 

Use with Ultra Streptavidin Detection Kit (BioLegend Cat # 929501) or (BioLegend Cat # 929401)

Positive control: Normal human skin

 

Protocol Steps


  1. Clear Slides: Removes paraffin and hydrates the tissue
    Note: If using frozen sections, allow slides to come to room temperature for 15 minutes & proceed to step (F) only

    A. Xylene - 5 minutes in each of (3) different 250 mL containers
    B. 100% alcohol - 5 minutes in each of (3) different 250 mL containers
    C. 95% alcohol - 3 minutes in (1) 250 mL container
    D. 70% alcohol - 3 minutes in (1) 250 mL container
    E. Water - 1 minutes in each of (3) different 250 mL containers
    F. H2O2 (3%) - 15 minutes in (1) 250 mL container  

  2. Rinse slides with lab grade water.
    Note: Lab grade filtered water such as injection grade, cell culture grade, Reverse Osmosis De-Ionization (RODI).  

  3. Antigen Retrieval

    A. 70% Formic Acid - incubate the slides for 20 minutes at room temperature. Note: This antigen retrieval step is harsh on the tissue. If using frozen sections reduce time to 5-10 minutes or omit if tissue falls off the slide.
    B. Rinse Slides with 1X PBS.
    C. Remove from microwave and allow slides to cool on the bench top for 10 minutes.
    D. Rinse slides with lab grade water.  

  4. Apply serum block for at least 5 minutes. Do not wash after this step.

  5. Blot off serum block.

  6. Apply primary antibody (see recommended dilution from datasheet).

  7. Incubate primary antibody for 60 minutes at room temperature.

  8. Rinse slides with 1X PBS.  

  9. Apply USA Linking reagent - 20 minutes incubation.

  10. Rinse slides with 1X PBS.  

  11. Apply Labeling Reagent - 20 minutes incubation

  12. Rinse with 1X PBS.  

  13. Apply chromogen - 5 minutes incubation. Dilute according to manufacturer's instructions.

    A. AEC Chromogen: 20 µL AEC chromogen + 1 mL AEC substrate buffer

  14. Rinse slides with lab grade water.  

  15. Coverslip

    A. Submerge slides in Mayer’s Hematoxylin for 30 seconds.
    B. Rinse under running lab grade water for 1 minute or until water is clear.
    C. Submerge slides in Bluing Reagent for 1 minute.
    D. Rinse under running lab grade water for 1 minute.
    E. Cover slip slide using Permanent Aqueous Mounting Medium.
    Note: do not use xylene based mount with AEC Chromogen as it will dissolve the chromogen.

 

General Tips & FAQ

  • Do I need to perform antigen retrieval on my formalin-fixed, paraffin-embedded samples prior to staining?
    • In most cases, this is true. Antigen retrieval helps both the accessibility of the antibody to the tissue and also counteracts the fixation effects on the recognized epitopes. Check the application references for use of the antibody in paraffin-embedded samples.
  • Can antibody X be used for immunohistochemistry? What concentration do I use?
    • Typical concentrations of monoclonal antibodies for use in IHC are from 5-25 µg/ml. Polyclonal antibodies can be used at a range of 1-10 µg/ml. While we do not test for IHC application in house, we will indicate on the datasheet if an antibody has been published for use in this application. In addition, you can do a literature search with the clone name and immunohistochemistry/paraffin/frozen to see what the protocol details are.
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