Chapter 23 In Situ Hyridization

Abstract

This chapter describes the various protocols that are developed for localizing gene transcripts in both sectioned and whole-mount Xenopus tissue. Because of its wide application, the methods used for in situ hybridization continue to evolve as protocols for hybridizing nucleic acid probes to tissue are refined and improved. Many of these improvements can be incorporated directly into the protocols used for in situ hybridization to frog tissue. Nonetheless, there are several special modifications that are made to the Xenopus protocols which are essential for success, particularly when the technique is applied to oocytes and early embryos. These modifications were introduced to solve the problems that stem from the high yolk content and unusually large size of oocytes, eggs, and cells in early embryos. Another special consideration in applying in situ hybridization to Xenopus is that the density of gene transcripts in oocytes, eggs, or cells in the early embryos is decreased by the displacement of the cytoplasm with yolk granules. This decrease in transcript density effectively reduces the hybridization signal in a situation where the detection of rare transcripts is already at the limit of the technique.