Sensitive two-color whole-mount in situ hybridizations using digoxygenin- and dinitrophenol-labeled RNA probes

摘要

Two-color whole-mount in situ hybridization is a powerful technique for comparing the spatial expression patterns of two genes in developing embryos or within a given adult tissue. Many current procedures use two genespecific antisense RNA probes, each incorporating a different chemical moiety that is recognized by a specific antibody. Typically, the first gene-specific RNA probe is labeled with digoxygenin (DIG) and the second with fluorescein or biotin. After hybridization to the embryos, each bound antisense probe is then visualized using a specific enzyme-conjugated antibody and a distinctive color reaction. These procedures work well in situations when both of the target genes are expressed at high levels but are less reliable for cases in which one or both of the genes are expressed at moderate or low levels. Often, these difficulties can be attributed to the differing sensitivities of the two hapten/antibody/color detection systems that are used.