title: In situ hybridization on whole-mount zebrafish embryos and young larvae.
abstract: The in situ hybridization uses a labeled complementary RNA strand to localize a specific mRNA sequence in a tissue. This method is widely used to describe the spatial and temporal expression patterns of developmentally regulated genes. Here we describe a technique that employs in vitro synthesized RNA tagged with digoxigenin uridine-5'-triphosphate (UTP) to determine expression of genes on whole-mount zebrafish embryos and young larvae. Following hybridization, the localization of the specific transcript is visualized immunohistochemically using an anti-digoxigenin antibody conjugated to alkaline phosphatase that hydrolyzes the 5-bromo-4-chloro-3-indolyl phosphate (BCIP) to 5-bromo-4-chloro-3-indole and inorganic phosphate. 5-Bromo-4-chloro-3-indole can be oxidized by nitro blue tetrazolium (NBT), which forms an insoluble dark blue diformazan precipitate after reduction.This protocol has been used for performing large-scale analyses of the spatial and temporal expression of the zebrafish genome, resulting in the description of more than 8,400 expression patterns that are available at the zebrafish information network (ZFIN.org) in the gene expression section.
authors.affiliation: Department of Cell Biology, University of Virginia, 1340 Jefferson Park Avenue, Charlottesville, VA, 22903, USA.
authors.forename: Bernard
authors.initials: B
authors.lastname: Thisse
authors.forename: Christine
authors.initials: C
authors.lastname: Thisse
mesh_terms: D000818:Animals; D004076:Digoxigenin / Q000031:analogs & derivatives; D004625:Embryo, Nonmammalian / Q000378:metabolism / Q000648:ultrastructure*; D005260:Female; D018507:Gene Expression Regulation, Developmental; D007150:Immunohistochemistry / Q000379:methods; D017403:In Situ Hybridization / Q000379:methods*; D007202:Indicators and Reagents; D007211:Indoles; D007814:Larva / Q000235:genetics / Q000648:ultrastructure*; D016133:Polymerase Chain Reaction / Q000379:methods; D012313:RNA / Q000032:analysis*; D015347:RNA Probes / Q000032:analysis / Q000235:genetics; D016707:Tissue Fixation / Q000379:methods; D014544:Uridine Triphosphate / Q000031:analogs & derivatives; D015027:Zebrafish / Q000196:embryology* / Q000235:genetics
chemical_list: D007202:Indicators and Reagents; D007211:Indoles; D015347:RNA Probes; C035455:5-bromo-4-chloro-3-indoxyl phosphate; D012313:RNA; D004076:Digoxigenin; D014544:Uridine Triphosphate