Nonradioactive in situ hybridization to xenopus tissue sections

Methods : a Companion to Methods in Enzymology
K ButlerJ B Gurdon

Abstract

We describe a protocol for the localization of specific messenger RNAs in Xenopus laevis embryo tissue sections using a nonradioactive detection method. After fixation, embryos are embedded in paraffin wax, sectioned, mounted on slides, and subjected to a series of prehybridization treatments which improve the accessibility of the probe to the target mRNA and reduce nonspecific binding. These treatments are followed by hybridization in situ with single-stranded antisense RNA probe generated by in vitro transcription and labeled with digoxigenin. The hybridization products are detected with preabsorbed alkaline phosphatase-coupled digoxigenin antibody and subsequently localized using a chromogenic substrate that generates a colored precipitate at the hybridization site. The nonradioactive in situ hybridization method we describe is reproducible and has a detection sensitivity akin to those methods that use antisense RNA probes labeled with radioisotopes; however, it is faster, safer, and easier to perform. Sectioning of prestained whole-mount X. laevis embryos does not always show the complete expression pattern of many genes, particularly those in deep endodermal structures, due to inadequate probe penetration. Therefore thorou...Continue Reading

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Citations

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