Immunodetection of RNA on ultra-thin sections incubated with polyadenylate nucleotidyl transferase.
A new method is described for locating RNA on ultra-thin sections. Sections of aldehyde-fixed, plastic-embedded cells were incubated in a medium containing polyadenylate nucleotidyl transferase (PnT) and biotinylated ATP. The labeled nucleotides bound to RNA at the surface of the ultra-thin sections were than visualized by an indirect immunogold labeling technique. The resulting labeling pattern was dependent on the presence of divalent cations in the PnT medium. The method revealed with great precision the specific RNA-containing structures within Ehrlich tumor cells. The method is applicable to Epon sections. However, the labeling intensity varies according to the fixation used. Best results were obtained on acetylated cell sections. The method can be combined with EDTA regressive staining. The in situ PnT method provides a very useful tool for pinpointing the precise location of RNA within biological material at the ultrastructural level.
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Laboratory of Cellular and Tissular Biology, University of Liège, Pitteurs, Belgium.
D000818:Animals; D002286:Carcinoma, Ehrlich Tumor / Q000737:chemistry; D007150:Immunohistochemistry; D051379:Mice; D008810:Mice, Inbred C57BL; D016253:Microscopy, Immunoelectron; D011062:Polynucleotide Adenylyltransferase; D012313:RNA / Q000032:analysis*; D012334:RNA, Neoplasm / Q000032:analysis
D012334:RNA, Neoplasm; D012313:RNA; D011062:Polynucleotide Adenylyltransferase