title: Detection and signal amplification in zebrafish RNA FISH.
abstract: In situ hybridization (ISH) has become an invaluable tool for the detection of RNA in cells, tissues and organisms. Due to improvements in target and signal amplification and in probe design remarkable progress has been made concerning sensitivity, specificity and resolution of chromogenic and fluorescent ISH (FISH). These advancements allow for exquisite cellular and sub-cellular resolution and for detecting multiple RNA species at a time by multiplexing. In zebrafish (F)ISH non-enzymatic and enzymatic amplification systems have been employed to obtain enhanced signal intensities and signal-to-noise ratios. These amplification strategies include branched DNA-based RNAscope and in situ hybridization chain reaction (HCR) techniques, as well as alkaline phosphatase (AP)- and horseradish peroxidase (PO)-based immunoassays. For practical application, we provide proven multiplex FISH protocols for AP- and PO-based visualization of mRNAs at high resolution. The protocols take advantage of optimized tyramide signal amplification (TSA) conditions of the PO assay and long-lasting high signal-to-noise ratio of the AP reaction, thereby enabling detection of less abundant transcripts.
authors.affiliation: Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, Stockholm, Sweden. Electronic address: giselbert.hauptmann@su.se.
authors.forename: Giselbert
authors.initials: G
authors.lastname: Hauptmann
authors.affiliation: Department of Biosciences and Nutrition, Karolinska Institutet, Huddinge, Sweden.
authors.forename: Gilbert
authors.initials: G
authors.lastname: Lauter
authors.affiliation: Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, Stockholm, Sweden.
authors.forename: Iris
authors.initials: I
authors.lastname: Söll
mesh_terms: D000469:Alkaline Phosphatase / Q000737:chemistry; D000818:Animals; D004625:Embryo, Nonmammalian / Q000378:metabolism / Q000648:ultrastructure*; D005456:Fluorescent Dyes / Q000737:chemistry; D020869:Gene Expression Profiling; D018507:Gene Expression Regulation, Developmental; D006735:Horseradish Peroxidase / Q000737:chemistry; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D012333:RNA, Messenger / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D059629:Signal-To-Noise Ratio; D000072760:Single Molecule Imaging / Q000379:methods*; D016707:Tissue Fixation / Q000379:methods; D014158:Transcription, Genetic; D014439:Tyramine / Q000737:chemistry; D015027:Zebrafish / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism
chemical_list: D005456:Fluorescent Dyes; D012333:RNA, Messenger; D006735:Horseradish Peroxidase; D000469:Alkaline Phosphatase; D014439:Tyramine
keywords: Colocalization analysis; Gene expression profiling; Multiplex fluorescent in situ hybridization; Non-enzymatic and enzymatic signal amplification; mRNA localization