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

  • Bernard Thisse
  • Christine ThisseEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1211)

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 

Notes

Acknowledgment

This work was supported by funds from the University of Virginia.

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Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  1. 1.Department of Cell BiologyUniversity of VirginiaCharlottesvilleUSA

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