Topological organization of Drosophila Hox genes using DNA fluorescent in situ hybridization

DNA fluorescent in situ hybridization (FISH) is the method of choice to study genomic organization at the single-cell level. It has been recently used to study the topological organization of the homeotic bithorax complex (BX-C) in Drosophila as well as to describe long-range genomic interactions between the BX-C and the Antennapedia complex (ANT-C), in addition to other genomic loci. Coupled with immunofluorescence, FISH can be used to study the relative positioning of homeotic genes with nuclear subcompartments, such as Polycomb-group (PcG) bodies, transcription factories, or the nuclear lamina. Here, we describe two multicolor 3D-FISH protocols; one for whole mount Drosophila embryos or larval discs and one for Drosophila-cultured cells. Both methods can be applied to any single copy locus of interest and are compatible with immunostaining (FISH-I).