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ZFIN ID: ZDB-PUB-140915-2
In Situ Hybridization on Whole-Mount Zebrafish Embryos and Young Larvae
Thisse, B., Thisse, C.
Date: 2014
Source: Methods in molecular biology (Clifton, N.J.)   1211: 53-67 (Chapter)
Registered Authors: Thisse, Bernard, Thisse, Christine
Keywords: none
MeSH Terms:
  • Animals
  • Digoxigenin/analogs & derivatives
  • Embryo, Nonmammalian/metabolism
  • Embryo, Nonmammalian/ultrastructure*
  • Female
  • Gene Expression Regulation, Developmental*
  • Immunohistochemistry/methods
  • In Situ Hybridization/methods*
  • Indicators and Reagents
  • Indoles
  • Larva/genetics
  • Larva/ultrastructure*
  • Polymerase Chain Reaction/methods
  • RNA/analysis*
  • RNA Probes/analysis
  • RNA Probes/genetics
  • Tissue Fixation/methods
  • Uridine Triphosphate/analogs & derivatives
  • Zebrafish/embryology*
  • Zebrafish/genetics
PubMed: 25218376 Full text @ Meth. Mol. Biol.
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 ( in the gene expression section.