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The in situ hybridization (ISH) technique, as applied to electron microscopic detection of RNAs, was evaluated for ultra-thin cryosections of cultured rat fibroblasts (rat 9G). Experimental variables to balance penetration of detection reagents and preservation of ultrastructural morphology included various strengths of aldehyde fixation and pepsin treatment. We performed ISH for 28S ribosomal RNA (rRNA) followed by ultra-small colloidal gold immunocytochemistry and silver enhancement. An acceptable balance for 28S rRNA ISH detection was obtained using mild cross-linking fixation followed by treatment with a relative high concentration of pepsin for a short time. The ISH method presented in this study was compatible with immunocytochemical detection of protein as demonstrated by double-labeling experiments.

Evaluation of pepsin treatment for electron microscopic RNA in situ hybridization on ultra-thin cryosections of cultured cells