Highly sensitive immunodetection of DNA on sections with exogenous terminal deoxynucleotidyl transferase and non-isotopic nucleotide analogues.
A new method is described for locating DNA on ultra-thin sections. Sections of aldehyde-fixed, plastic-embedded cells were incubated in a medium containing terminal deoxynucleotidyl transferase (TdT) and various non-isotopic nucleotide analogues. The labeled nucleotides bound to the surface of ultra-thin sections were then visualized by an indirect immunogold labeling technique. The resulting labeling pattern was strongly dependent on the divalent cation used in the TdT medium. The method revealed with great precision the specific DNA-containing structures within Ehrlich tumor cells, even where DNA was present in very low amounts. The method is compatible with all usual fixation and embedding procedures and can be combined with cytochemical methods. The in situ TdT method provides a very useful tool for pinpointing the precise location of DNA within biological material at the ultrastructural level.
Thiry
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Laboratoire de Biologie Cellulaire et Tissulaire, Université de Liège, Belgique.
D000818:Animals; D001973:Bromodeoxyuridine; D002286:Carcinoma, Ehrlich Tumor / Q000648:ultrastructure*; D002843:Chromatin / Q000648:ultrastructure; D004247:DNA / Q000032:analysis*; D004253:DNA Nucleotidylexotransferase; D004273:DNA, Neoplasm / Q000032:analysis*; D006652:Histological Techniques; D007202:Indicators and Reagents; D051379:Mice; D008810:Mice, Inbred C57BL; D016253:Microscopy, Immunoelectron / Q000379:methods; D009685:Nuclear Envelope / Q000648:ultrastructure; D009711:Nucleotides / Q000378:metabolism; D014407:Tumor Cells, Cultured
D002843:Chromatin; D004273:DNA, Neoplasm; D007202:Indicators and Reagents; D009711:Nucleotides; D004247:DNA; D004253:DNA Nucleotidylexotransferase; D001973:Bromodeoxyuridine