One-, two-, and three-color whole-mount in situ hybridization to Drosophila embryos

This article contains detailed protocols for the localization of mRNA trans cripts within whole Drosophila embryos. The procedures are based on the use of digoxigenin-, fluorescein-, and biotin-labeled antisense RNA probes for nonradioactive detection of transcripts. The labels are visualized in situ by differently colored water-insoluble precipitates using alkaline phospha tase- or beta -galactosidase-based immunoassays. First, a basic method is d escribed that allows detection of transcript distribution(s) of one or more genes using the same color precipitate. Second, a sequential alkaline phos phatase detection method is presented that permits the visualization of two or three independent transcript patterns in multiple colors in the same em bryo. Third, a shortened two-color in situ hybridization protocol is provid ed that employs a combination of beta -galactosidase and alkaline phosphata se colorimetric reactions for differential detection. The two-color in situ hybridization methods work equally well In Drosophila and zebrafish embryo s and may therefore also be adaptable to other species. (C) 2001 Academic P ress.