Multicolor Whole-Mount In Situ Hybridization

In situ hybridization is a procedure that allows the detection of the site(s) of transcription of a given gene at a cellular level within an entire organism. Since its introduction into developmental biology (1 ), this procedure has become an indispensable tool to investigate gene expression; initial protocols employed tritiated probes. These procedures required very long exposure times and allowed the detection of transcripts only on tissue sections. The method was drastically improved by the use of nonradioactive digoxigenin-labeled probes. This allowed the detection of gene transcription also in whole-mounted Drosophila embryos (2 ). This enhanced technique allowed to obtain results within a few days instead of weeks. The Drosophila protocol was quickly adapted to embryos of vertebrate model organisms that are widely used in developmental biology like Xenopus (3 ), zebrafish (4 ) and mice (5 ).