Double in situ hybridization techniques in zebrafish

Over recent years many genes expressed in zebrafish embryos have been cloned and a first step in their analysis is the determination of the spatial and temporal expression patterns of their transcripts by in situ hybridization. It is often necessary to relate the expression pattern to that of other genes expressed at the same period of development. Transcripts from different genes may be expressed in complementary or overlapping domains. The pattern of expression of different genes can be related by performing in situ hybridization on consecutive sections of tissue but it is also possible to perform hybridizations with two probes and to visualize the signals separately in the same tissue. Occasionally it is also possible to combine in situ hybridization with immunocytochemistry to localize tissue-specific antigens. Methods developed for performing these types of analyses in zebrafish are described in this article.