Fluorescence in situ hybridization.docVIP

Fluorescence in situ hybridization FISH Fluorescent in situ hybridization FISH is a powerful technique that uses non-toxic fluorescent DNA probes to target any given sequence within a nucleus, resulting in colored signals that are detected with a fluorescence microscope. It circumvents the need for tumor cell-culture fresh or paraffin-embedded interphase nuclei can be analyzed directly , and provides researchers with a quick and precise screening method over large quantities of cells. Standard FISH techniques with chromosome or locus-specific probes can identify rearrangements too subtle or too complex to be disclosed by classical chromosome banding, with the downside of revealing only those aberrations one tests for. This is therefore not suitable for the initial screening of samples with an unknown genetic background. However, recent developments have made available FISH-based techniques with much wider specificity than the original method, such as multicolor-FISH M-FISH and comparative genomic hybridization CGH , which our group has extensive experience in performing. More specifically, M-FISH uses a pool of whole-chromosome painting probes with different fluorochrome combinations that result in specific color patterns for each human chromosome. It can detect complex inter-chromosomal rearrangements or changes involving segments too small or too similarly banded to be adequately described by standard banding, with the downside of requiring metaphases i.e., tumor cell culture to be performed.  M-FISH metaphase spread of colon cancer cell line V9P Kleivi et al. 2004 Method overviewInterphase FISH IP-FISH . For fresh samples, tissue is disaggregated with scalpel and/or enzymatic solution and the isolated cells are treated with a hypotonic solution to burst the plasma membranes and free the nuclei , fixed in methanol and dropped onto glass slides. A quick pre-treatment procedure is performed using a warm solution of 2xSSC to remove cytoplasm b