Nonradioactive in situ hybridization to Xenopus tissue sections

We describe a protocol for the localization of specific messenger RNAs in X enopus laevis embryo tissue sections using a nonradioactive detection metho d. 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 nonspe cific: binding. These treatments are followed by hybridization in situ with single-stranded antisense RNA probe generated by in vitro transcription an d labeled with digoxigenin. The hybridization products are detected with pr eabsorbed alkaline phosphatase-coupled digoxigenin antibody and subsequentl y localized using a chromogenic substrate that generates a colored precipit ate at the hybridization site. The nonradioactive in site hybridization met hod we describe is reproducible and has a detection sensitivity akin to tho se methods that use antisense RNA probes labeled with radioisotopes; howeve r, it is faster, safer, and easier to perform. Sectioning of prestained who le-mount X. laevis embryos does not always show the complete expression pat tern of many genes, particularly those in deep endodermal structures, due t o Inadequate probe penetration. Therefore thorough analysis of gene express ion patterns often requires in situ hybridization on presectioned material whereby probe has equal accessibility to all tissue. (C) 2001 Academic Pres s.