Three-dimensional laser-scanning confocal microscopy of in situ hybridization in the skin

In situ hybridization is an important tool in molecular and developmental biology to detect specific nucleic acid sequences (either mRNA or DNA) within cells. This technique is especially applicable to tissue sections since it provides information about the spatial distribution of DNA or mRNA sequences. However, previous studies utilizing in situ hybridization in the skin were hampered by a high degree of nonspecific background, which has made interpretation of the results difficult. In this paper, we demonstrate how refinements in in situ hybridization techniques, combined with laser-scanning confocal microscopy, significantly reduce nonspecific background and produce improved resolution of in situ hybridization in skin specimens. The sensitive detection method of laser-scanning confocal microscopy allows three- dimensional localization of S³⁵ radioactive-labeled riboprobes within the emulsion of specimens, which is not possible with conventional bright or dark field light microscopy.