Protocol

In Situ Hybridization Protocols

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

Date:

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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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.

Key words

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