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A general method for the ultrastructural localization of intracellular proteins and antigens by immunoferritin techniques has been developed. The method involves direct staining of ultrathin sections of mildly glutaraldehyde-fixed and frozen tissues cut by means of a cryo-ultramicrotome. Bovine pancreatic sections were cut, mounted on grids, and stained with ferritin-rabbit antibovine RNase conjugates. After negative staining with 0.2% phosphotungstic acid, electron micrographs revealed specific labeling of all of the zymogen granules and the cisternae of the rough endoplasmic reticulum. No significant labeling was seen in the nucleus, mitochondria, or cell sap regions. The observation that no significant labeling was found in any region of rat pancreatic sections was consistent with the fact that rat RNase is immunologically non-crossreactive with bovine RNase. In addition, the labeling seen in bovine pancreas was completely absent if the sections were first incubated with free antibody. The method used here avoids prolonged fixation, dehydration, and other harsh chemical or physical treatments, and should extend the usefulness of immunoferritin techniques to the intracellular localization of many protein antigens beyond previously available methods.

proceedings of the national academy of sciences of the united states of america

Immunoferritin Localization of Intracellular Antigens: The Use of Ultracryotomy to Obtain Ultrathin Sections Suitable for Direct Immunoferritin Staining