Nonradioactive in situ hybridization to Xenopus tissue sections

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 site 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 thorough analysis of gene expression patterns often requires in situ hybridization on presectioned material whereby probe has equal accessibility to all tissue. (C) 2001 Academic Press.