title: One-, two-, and three-color whole-mount in situ hybridization to Drosophila embryos.
abstract: This article contains detailed protocols for the localization of mRNA transcripts 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 phosphatase- or beta-galactosidase-based immunoassays. First, a basic method is described that allows detection of transcript distribution(s) of one or more genes using the same color precipitate. Second, a sequential alkaline phosphatase detection method is presented that permits the visualization of two or three independent transcript patterns in multiple colors in the same embryo. Third, a shortened two-color in situ hybridization protocol is provided that employs a combination of beta-galactosidase and alkaline phosphatase colorimetric reactions for differential detection. The two-color in situ hybridization methods work equally well in Drosophila and zebrafish embryos and may therefore also be adaptable to other species.
authors.affiliation: Institut für Biologie I (Zoologie), Albert-Ludwigs-Universität Freiburg, Hauptstrasse 1, Freiburg im Breisgau, D-79104, Germany.
authors.forename: G
authors.initials: G
authors.lastname: Hauptmann
mesh_terms: D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D001710:Biotin / Q000494:pharmacology; D004076:Digoxigenin / Q000494:pharmacology; D004330:Drosophila; D004625:Embryo, Nonmammalian / Q000378:metabolism*; D017403:In Situ Hybridization / Q000379:methods*; D015335:Molecular Probes / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D001616:beta-Galactosidase / Q000378:metabolism
chemical_list: D015335:Molecular Probes; D012333:RNA, Messenger; D001710:Biotin; D000469:Alkaline Phosphatase; D001616:beta-Galactosidase; D004076:Digoxigenin