The detection of transcript distribution throughout a fixed tissue is a major step in studying the transcriptional activity of target genes and their function. In situ hybridization specifically detects the spatial distribution of RNA transcripts using an antisense RNA probe. This protocol describes the preparation of digoxigenin-labeled antisense RNA probes and their hybridization to complementary mRNA sequences; expression can then be localized using an antibody against digoxigenin conjugated to a chromogenic enzyme. In ants, this method can be applied to visualize cell populations of interest among other populations in a tissue, such as insect ovaries, or in the whole organism, such as insect embryos. Specific markers (e.g., genes known to be expressed in particular clusters of cells) can be cloned and used as probes to study the distribution and development of germline cells (e.g., nanos, vasa), neurons (repo), or limb structures (e.g., distal-less). Various markers might also allow the study of oogenesis (nanos, par-1, oskar), segmentation in the embryo (e.g., engrailed, wingless), or other developmental processes.
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