Evaluation of pepsin treatment for electron microscopic RNA in situ hybridization on ultra-thin cryosections of cultured cells |
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| Authors: | Merryn V. E. Macville Annette G. M. Van Dorp Roeland W. Dirks Jack A. M. Fransen Anton K. Raap |
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| Affiliation: | (1) Department of Cytochemistry and Cytometry, Sylvius Laboratories, University of Leiden, Wassenaarseweg 72, NL2333 AL Leiden, The Netherlands;(2) Department of Electron Microscopy, University of Leiden, Leiden, The Netherlands;(3) Present address: Department of Dermatology, Academic Hospital Leiden, Leiden, The Netherlands;(4) Present address: Department of Cell Biology and Histology, University of Nijmegen, Nijmegen, The Netherlands |
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| Abstract: | 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. |
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