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:

Materials:

• 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.

Steps:

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.