Fluorescent in situ hybridization protocols in Drosophila embryos and tissues

Fluorescent in situ hybridization (FISH) is the standard method for visualizing the spatial distribution of RNA. While traditional histochemical RNA detection methods suffered from limitations in resolution or sensitivity, the recent development of peroxidase-mediated tyramide signal amplification (TSA) provides strikingly enhanced sensitivity and subcellular resolution. In this chapter, we describe optimized FISH protocols for Drosophila embryos and tissues utilizing TSA, either for single genes or in a high-throughput format, which greatly increase the sensitivity, consistency, economy and throughput of the procedure. We also describe variations of the method for RNA-RNA and RNA-protein co-detection. Key words Drosophila melanogaster, embryos and tissues, fluorescent in situ hybridization, FISH, tyramide signal amplification, single and double labeling, RNA-protein co-staining. 1