Whole-Mount In Situ Hybridization of Xenopus Embryos.

Abstract

Historically, techniques to analyze the localized distribution of mRNAs during development were performed on sectioned embryos using radioactively labeled riboprobes. The processing of the tissues and the use of emulsion autoradiography were laborious and time-consuming, leading to the development of more direct approaches. The nonradioactive whole-mount in situ hybridization method was first introduced in Drosophila embryos, and later adapted to Xenopus embryos for abundant transcripts such as muscle actin. Since then, the technique has been improved and is now broadly used for the spatial detection of even less abundant transcripts in Xenopus The technique has been especially powerful in the analysis of changes in gene expression in embryos manipulated by mRNA or antisense oligonucleotides microinjection, and in animal cap explants exposed to exogenous factors. The protocol described here provides an excellent signal-to-noise ratio for most labeled probes. It also is relatively high-throughput: With a little practice, approximately 50 samples can easily be processed simultaneously.