Pre-treatment for the new histologic-based FISH assay is similar to the antigen retrieval process in IHC staining. Several vendors 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 we use pre-treatment in FISH. The Formalin fixation is typically used to fix tissue before paraffin embedding cross-link proteins. The pre-treatment or antigen retrieval step breaks down the aldehyde cross-links and allows access to either the antibody in IHC or the probe in FISH to penetrate 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 the temperature inside the 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 the lid and allow the 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 and let them sit for 3 minutes at room temperature (20–25°C).
- Replace the wash buffer and soak sections for another 3 minutes.
