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The page below is a sample from the LabCE course Technical Competence in Paraffin-Based Fluorescence In Situ Hybridization (FISH). Access the complete course and earn ASCLS P.A.C.E.-approved continuing education credits by subscribing online.

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Differences in Pre-treatment

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:
  1. Heat the pre-treatment solution to 95-99° C. Measure the temperature inside the container to ensure accuracy.
  2. Immerse the room temperature, deparaffinized sections into the preheated solution.
  3. 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).
  4. Remove the entire container with slides from the water bath.
  5. Remove lid and allow slides to cool in the pre-treatment solution for 15 minutes at room temperature (20-25° C).
  6. Transfer the slides to a container with diluted wash buffer for 3 minutes at room temperature (20-25° C).
  7. Replace wash buffer and soak sections for another 3 minutes.