Pre-treatment for the new histologic-based FISH assay is very similar to the antigen retrieval process used in IHC staining. There are several vendors that produce paraffin pre-treatment kits for FISH staining. A citrate buffer at pH 6.0 is often used in IHC for antigen retrieval. The purpose of antigen retrieval in IHC is similar to the reason that we use pre-treatment in FISH. The formalin fixation typically used to fix tissue prior to paraffin embedding cross-links proteins. The pre-treatment step, or antigen retrieval step, breaks down the aldehyde cross-links and allows access of either the antibody in IHC or the probe in FISH to be able to penetrate into the cell.
To perform the pre-treatment section of the assay:
- Heat the pre-treatment solution to 95-99° C. Measure the temperature inside the container to ensure accuracy.
- Immerse the room temperature, deparaffinized sections into the preheated solution.
- Re-check temperature inside pre-treatment container and incubate for 10 minutes at 95-99° C. Do not start the timer until the solution temperature inside the container is appropriate (95-99° C).
- Remove the entire container with slides from the water bath.
- Remove lid and allow slides to cool in the pre-treatment solution for 15 minutes at room temperature (20-25° C).
- Transfer the slides to a container with diluted wash buffer for 3 minutes at room temperature (20-25° C).
- Replace wash buffer and soak sections for another 3 minutes.
