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METHOD FOR ENHANCEMENT OF ELECTRON MICROSCOPIC VISUALIZATION OF EMBEDDED ANTIGEN BY BRIDGING OSMIUM TO URANIUM ANTIBODY WITH THIOCARBOHYDRAZIDE

LUDWIG A. STERNBERGER 1, JACOB S. HANKER 1, EDWARD J. DONATI 1, JOHN P. PETRALI 1, and ARNOLD M. SELIGMAN 1

1 Pathology Branch, Medical Research Laboratories, Edgewood Arsenal, Maryland, and the Departments of Surgery, Sinai Hospital of Baltimore, Inc., and The Johns Hopkins University School of Medicine, Baltimore, Maryland

Thiocarbohydrazide (TCH), a bidentate ligand, capable of bridging osmium to osmium in ultrathin sections (OTO method), was shown to possess the additional property of binding osmium to uranium-labeled antibody. On sections in which acrolein-fixed antigen had been localized for electron microscopy by uranium antibody, application of TCH followed by osmication (T-O procedure) greatly enhanced the specific contrast afforded. In addition, the T-O procedure yielded better resolution and better stability to the electron beam than the immunouranium technique employed without this intensification. Even osmium tetroxide-fixed tissue could be used, providing thiosemicarbazide (TSC) was employed to block the later reaction of TCH with tissue-bound osmium and to limit, thereby, the ligation of TCH to the uranium-labeled antibody.

Submitted on June 28, 1966


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