Whole-Mount Fluorescence In Situ Hybridization and Antibody Staining of Drosophila Embryos

Abstract

Whole-mount RNA in situ hybridizations with digoxigenin-conjugated probes and alkaline phosphatase biochemistry have been used widely for many years to map expression pattern domains in the Drosophila embryo. To capitalize on the number of molecular markers in the central nervous system (CNS) and to enable expression analysis at the single-cell level, fluorescence in situ hybridization procedures are becoming standard. This protocol describes methods for the simultaneous detection of RNA and protein using fluorescence in Drosophila embryos. It uses the tyramide signal amplication (TSA) system from PerkinElmer to amplify a horseradish peroxidase (HRP) signal. By combining this technology with an HRP-conjugated antidigoxigenin antibody, we can detect standard antidigoxigenin RNA probes fluorescently.