High-resolution fluorescent in situ hybridization of Drosophila embryos and tissues

INTRODUCTIONFluorescent in situ hybridization (FISH) is commonly used to analyze the three-dimensional distribution of RNAs in intact embryos and tissues. Tyramide signal amplification (TSA) significantly increases the sensitivity and resolution of FISH probe signals. This protocol includes optimized TSA-FISH procedures for Drosophila embryos, ovaries, and larval tissues. Instructions are given for the preparation of RNA probes, the collection and fixation of tissues, and the hybridization and TSA-mediated detection of probes, including options for high-throughput processing in 96-well plates. Variations of the procedure for RNA-RNA and RNA-protein costaining are also described.