Fluorescence in situ hybridization on formalin-fixed and paraffin-embedded tissue: optimizing the method

Fluorescence in situ hybridization (FISH) is widely used to study numerical and structural genetic abnormalities in both metaphase and interphase cells. The technique is based on the hybridization of labeled probes to complementary sequences in the DNA or RNA of the cells. Interphase FISH is most often applied on cytologic material such as hematologic smears or imprints, but the method is also used to study genetic changes in tissue sections when morphology is important or when cytologic material is not available. In cases in which the presence of intact nuclei is of importance, such as quantitation of signals as in triploidy, it is possible to isolate nuclei from paraffin-embedded tissue. However, using formalin-fixed paraffin-embedded tissue, either in thin sections or as isolated nuclei, one encounters a range of technical problems, paralleling those met in immunohistochemistry. Variations in time lapse between removal of tissue and fixation, duration of fixation, enzymatic pretreatment, hybridization conditions, and posthybridization washing conditions are important factors in the hybridization. In this study, we have listed the results of a systematic approach to improve FISH on isolated nuclei and tissue sections from formalin-fixed, paraffin-embedded tissue.