IMPROVED RESOLUTION OF FIBRONECTIN MESSENGER-RNA EXPRESSION IN THE INNER-EAR USING LASER-SCANNING CONFOCAL MICROSCOPY

We describe a modified in sim hybridization protocol for localizing an d quantifying fibronectin gene expression at the cellular level in par affin sections of rat temporal bone. When combined with a novel analyt ical approach using laser scanning confocal microscopy (LSCM), this pr otocol significantly improved the resolution, sensitivity, and specifi city of existing procedures for evaluating fibronectin synthesis in de veloping inner ear, For simultaneous viewing of cochlear anatomy and t he autoradiographic signal, transmitted light images of the cochlea we re collected separately from LSCM reflected light images of the autora diographic silver grains and then the two images were electronically m erged. Within the first 2 mu m below the surface of the emulsion, silv er grains were clustered specifically over hybridized cells. In contra st, nonspecific silver grain development (i.e., background noise) was confined primarily to the lower 5 mu m of the emulsion adjacent to the tissue section. Limiting the volume of the emulsion examined in the L SCM analysis, i.e., restricting the range of optical sectioning to the first 2 mu m below the surface of the emulsion, effectively minimized nonspecific background noise and maximized the specificity of the hyb ridization signal. The improvements offered by the described methodolo gical approaches are equally appropriate for non-calcified tissues.