Fluorescent in situ hybridization (FISH) of mitotic chromosomes from Drosophila larval brain

The fluorescent in situ hybridization (FISH) technique permits fine mapping of both middle and highly repetitive DNA sequences along Drosophila melanogaster heterochromatin. Best results are obtained when this technique is coupled with DAPI staining and digital recording of fluorescent signals. For example, if digital images of the FISH signals and DAPI fluorescence are detected separately using a charge-coupled device (CCD) camera, they can then be pseudocolored and merged using suitable computer programs. This allows precise overlapping of the DAPI banding (which is identical to the Hoechst 33258 banding) and the FISH signals, facilitating the assignment of the repetitive sequence under study to specific regions of the cytological map of D. melanogaster heterochromatin. This article describes FISH procedures that are routinely used with larval brain squashes, including preparation of slides, preparation of biotin- and digoxigenin-labeled probes, hybridization, and detection.