One of the most critical steps in the fluorescence in situ hybridization (FISH) assay for formalin-fixed, paraffin-embedded (FFPE) tissue is the pre-treatment step.
The reason the pre-treatment step is required is because formalin fixation results in the cross-linkage of proteins. The purpose of the pre-treatment step is to break these linkages, exposing the genes and allowing penetration of the probes. The pre-treatment step must also maintain the morphology of the tissue and the integrity of the DNA in the cells of interest.
Pre-treatment strategies have typically used either a sodium thiocyanate solution or a citric acid buffer. Both solutions are commonly used in clinical laboratories. The historical standard FISH method presented in this course uses a sodium thiocyanate solution and the second method presented in this course uses the citric acid solution.
Sodium thiocyanate is much harsher on tissue/cells than sodium citrate.
