Fluorescent in situ hybridization protocols in Drosophila embryos and tissues.
Fluorescent in situ hybridization is the standard method for visualizing the spatial distribution of RNA. Although traditional histochemical RNA detection methods suffered from limitations in resolution or sensitivity, the recent development of peroxidase-mediated tyramide signal amplification provides strikingly enhanced sensitivity and subcellular resolution. In this chapter, we describe optimized fluorescent in situ hybridization protocols for Drosophila embryos and tissues utilizing tyramide signal amplification, either for single genes or in a high-throughput format, which greatly increases the sensitivity, consistency, economy, and throughput of the procedure. We also describe variations of the method for RNA-RNA and RNA-protein codetection.
Lécuyer
Eric
E
Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, ON, Canada.
Parthasarathy
Neela
N
Krause
Henry M
HM
D000818:Animals; D001710:Biotin / Q000737:chemistry; D015509:Developmental Biology / Q000379:methods*; D004331:Drosophila melanogaster / Q000196:embryology* / Q000378:metabolism*; D004625:Embryo, Nonmammalian; D005821:Genetic Techniques; D006824:Hybridization, Genetic; D017404:In Situ Hybridization, Fluorescence / Q000295:instrumentation* / Q000379:methods*; D009693:Nucleic Acid Hybridization; D016376:Oligonucleotides, Antisense / Q000737:chemistry; D009195:Peroxidase / Q000378:metabolism; D012313:RNA / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism
D016376:Oligonucleotides, Antisense; D012333:RNA, Messenger; D012313:RNA; D001710:Biotin; D009195:Peroxidase