Immunohistochemistry (IHC) Protocol

Immunohistochemistry (IHC) Protocol

Immunohistochemistry (IHC) is a technique used to identify specific antigens in tissues by using specific antibodies that bind to them. Here’s a simple protocol:


  • Formalin-fixed paraffin-embedded (FFPE) tissue sections.
  • Xylene.
  • Series of graded alcohol.
  • Target retrieval solution.
  • Hydrogen peroxide.
  • Blocking solution.
  • Primary antibody, specific for the target antigen.
  • Biotinylated secondary antibody.
  • Streptavidin-HRP (horseradish peroxidase) and DAB (diaminobenzidine) chromogenic substrate or its equivalent.
  • Hematoxylin for counterstaining.
  • Slides and coverslips.
  • Mounting medium.


  1. Deparaffinization and rehydration:
    Soak slides in xylene for 10 minutes (2 changes), then in a series of graded alcohol for rehydration (100%, 95%, 80%, and 70%), each for 10 minutes.
  2. Antigen retrieval:
    Immerse slides in target retrieval solution and heat (in a microwave or pressure cooker) to unmask the antigens. Allow to cool down at room temperature. Rinney thoroughly with distilled water.
  3. Peroxidase block:
    To block endogenous peroxidase activity, incubate slides in a hydrogen peroxide solution for 10-15 minutes.
  4. Blocking:
    Apply blocking solution to reduce non-specific antibody binding.
  5. Primary antibody incubation:
    Incubate tissue with the primary antibody. Incubation times will vary based on the antibody (usually overnight at 2-8℃ or 1-2 hours at room temperature).
  6. Secondary antibody incubation:
    After washing off the primary antibody, incubate it with the biotinylated secondary antibody. Skip this step when using a directly conjugated primary antibody.
  7. DAB incubation:
    Apply a streptavidin-HRP complex, followed by DAB, which produces a brown colour at the antigen site.
  8. Counterstaining (optional):
    Apply hematoxylin to provide a blue counterstain to the brown DAB.
  9. Dehydration:
    Dehydrate slides with graded alcohol (reverse sequence as before) and then in xylene.
  10. Mounting:
    Mount with a coverslip using a mounting medium.

NOTE: This is a general protocol; products and incubation times can vary. Be sure to follow the specific instructions provided with your antibodies and reagents. Always wear appropriate personal protective equipment (PPE) for handling all chemicals.

Dr AF Saeed

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