As indicated in the preceding chapter, few techniques have had such a major impact on progress in the field of developmental biology as in situ hybridization of labeled RNA or DNA probes to detect specific mRNAs in embryonic tissues. Initially, the technique was performed on sections of fixed material. However, this has largely been superseded by the development of techniques for hybridization to RNA in intact embryos. The latter approach involves less effort, and has the additional advantages that a complete temporal study in all tissues can readily be undertaken in one or two experiments and that spatial and temporal changes in gene expression are more readily appreciated (see, e.g., Fig. 1A-C,F). The following procedure is suitable for hybridization to embryos with RNA probes (riboprobes) derived from DNA sequences from the same species and, with slight modification, is frequently suitable for use with probes from other species. This protocol (1-3) is a modified version of that of Wilkinson (4), and we have included procedures for "two-color" in situ hybridization, which allows the expression of two genes to be examined in the same embryo (see Fig. 1F). Protocols for the sectioning of material subsequent to in situ hybridization (see Fig. 1D,E) are also included.

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