Basic FISH Techniques and Troubleshooting

Fluorescence in situ hybridization (FISH) technology permits the detection of specific nucleic acid sequences in morphologically preserved chromosomes, cells, and tissue. The unambiguous detection of structural or copy number changes of whole chromosomes or chromosome specific regions is an important prognostic and predictive factor in human disease. The utility of FISH in chromosome analysis is divided into metaphase and interphase applications. This distinction is manifested primarily in the preparation of the cells prior to pretreatment and hybridization. The culturing of cells for metaphase analysis is identical to those used in standard karyotyping. FISH is particularly advantageous in samples of low cellularity, or containing a mixed population of cells. A wide variety of sample types are amendable to FISH making it particularly valuable in the study of archival material, such as formalin-fixed paraffin-embedded tissues. However, the success of probe hybridization is dependent on the optimal preparation of sample. In interphase applications, the efficiency of probe hybridization is greatly enhanced by treating the cell or tissue preparation with a variety of reagents that permeabilize the target while preserving important morphological features. Additionally, pretreatment will reduce the background autofluorescence of cells and tissue. A thorough understanding of the processes behind sample pretreatment will provide insight in troubleshooting efforts. Once the DNA in the sample is made available for hybridization, the conditions for optimal signal involves far fewer permutations. The purpose of this chapter is to review the salient features of FISH with respect to sample preparation and hybridization.