The design and generation of probes for in situ hybridization

Abstract The design and generation of probes for in situ hybridization (ISH) are critical determinants for the successful outcome of every experiment. It is necessary to use a labelled hybridization probe to detect the mRNA of interest in the target tissue section. There is no concern that nuclear DNA might also hybridize, because the double-stranded DNA is not denatured and thus not available for hybridization in mRNA ISH protocols. A variety of probes can be used, and they can be synthesized in different ways. Most often, radiolabelled probes are used together with autoradiography, but new methods of detecting non-isotopic probes are becoming widely used (see section on probe labelling below).