Ultrastructural antibody localization of alpha2-macroglobulin in membrane-limited vesicles in cultured cells.

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Abstract
A procedure for localizing intracellular antigens in cultured cells, using peroxidase-labeled antibodies, although useful, did not preserve the morphology of membranes and the location of the peroxidase reaction product was difficult to establish. Major improvements were made on the basic technique that markedly enhance the quality of localization and of morphology. Saponin is used to permeabilize membranes without destroying their morphology. The amount of reaction product is enhanced with a peroxidase-antiperoxidase label. The clarity of morphologic detail and contrast of reaction product density are increased by using postsectioning staining with the osmium/thiocarbohydrazide/osmium and uranyl acetate/lead citrate procedures. This technique was applied to the ultrastructural localization of .alpha.2-macroglobulin and demonstrated that it is localized in membrane-limited vesicles [of mouse 3T3-4 fibroblasts]. This method was also used to improve the preservation of structures for localization by fluorescence microscopy.

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