Standard Fluorescence in situ Hybridization Procedures

Fluorescence in situ hybridization (FISH) allows nucleic acid sequences to be visualized inside fi xed cells or chromosomes. The method, based on the principle that nucleic acids can denature and renature, allows the detection of a known nucleotide sequence (probe), in a cell/chromosome, thanks to its ability to hybridize the complementary nucleic-acid sequence (target) preserved in situ. FISH probes specifi c for fi sh are not commercially available and must be homemade. Before the hybridization experiment, the DNA probe has to be directly or indirectly labelled with a fl uorochrome, so that the hybridization product can be revealed and visualized as a fl uorescent signal. In direct labelling, the fl uorochrome is directly bound to the DNA probe. In indirect labelling, a hapten (most frequently biotin or digoxigenin) is incorporated in the probe and immunochemically detected by a fl uorophore-tagged antibody. The labelled probe and the target sequence are denatured and then combined, so that their complementary DNA sequences are annealed. When the probe and target DNA belong to the same species, the FISH is referred as homologous, whereas it is heterologous when they are derived from different species.