In Situ Hybridization Protocols

Volume 1211 of the series Methods in Molecular Biology pp 53-67


In Situ Hybridization on Whole-Mount Zebrafish Embryos and Young Larvae

  • Bernard ThisseAffiliated withDepartment of Cell Biology, University of Virginia
  • , Christine ThisseAffiliated withDepartment of Cell Biology, University of Virginia Email author 

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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.

Key words

In situ hybridization RNA Digoxigenin Alkaline phosphatase Expression pattern Synexpression Zebrafish Embryos Larvae