We generally recommend to:
- Boil slides in 0.01M sodium citrate buffer (pH6) at 100°C for 15-20 minutes.
- Remove the slides from heat and allow them to stand at RT in buffer for 20 minutes
- Rinse twice with TBST for 5 minutes at RT.
We have attached our IHC-Paraffin and IF(IHC-Paraffin) protocols for reference. Read below for more information about Antigen Retrieval.
The paraffin fixation process generates protein crosslinks, which mask certain epitopes from antibody binding. The antigen retrieval step is designed to break these linkages before the tissue is blocked and probed with antibodies.
There are two primary methods of antigen retrieval:
Heat-mediated (Heat-induced epitope retrieval): uses a pressure cooker, microwave, or water bath; must be optimized for tissue type, as heat can dissociate some samples from microscope slide
Enzyme-mediated: uses a combination of citrate, Tris, HCl, and EDTA buffers to chemically break linkages
Note: Heat-mediated antigen retrieval cannot be performed on frozen sections. However, since most frozen IHC protocols call for alcohol-based fixation steps, they eliminate the chance for crosslinking and therefore skip the antigen retrieval step entirely.
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