Monitoring: IHC

Question 1

What are the steps used to do immunohistochemistry?

Answer 1

1. Collect biopsy specimens
2. Tissue preparation
3. Antigen retrieval: need to revert some of the chemical crosslinking and “retrieve” the epitopes before proceeding to IHC (by heat or enzymatic)
4. Antibody binding: primary antibody selection
5. Detection:
   a. Secondary antibodies targeted specifically to antibody molecules from animal species (e.g. X-a-Ra-IgG)
   b. Enzymatic reaction: horseradish peroxidase (HRP) converts 3,3’ Diaminobenzidine (DAB) into brown precipitate
6. Counterstain: enhances the contrast and facilitates observation of histological features. Hematoxylin is most often used

Question 2

What is possible with chromogenic multiplex immunohistochemistry?

Answer 2

With chromogenic multiplex immunohistochemistry, you can use multiple Abs with different colors and look at multiple antigens of interest.
You can only use up to 4 colors.
The staining is often quite weak.

Question 3

What is the difference in method between multiplex immunohistochemistry and normal immunohistochemistry?

Answer 3

Instead of adding DAB, you add a TSA reagent (different fluorophores).