ANTIGEN UNMASKING FOR IMMUNOELECTRON MICROSCOPY - LABELING IS IMPROVED BY TREATING WITH SODIUM ETHOXIDE OR SODIUM METAPERIODATE, THEN HEATING ON RETRIEVAL MEDIUM

To optimize the ultrastructural localization of immunoglobulin G in co rneal crystalloid deposits, we compared a range of antigen unmasking t echniques. A human corneal biopsy specimen was fixed in formalin, post -osmicated, and embedded in epoxy resin for electron microscopy. Thin sections were immunogold-labeled for IgG after treatment with sodium e thoxide or sodium metaperiodate. Sections mere also treated by heating them at 95 degrees C while they floated on water, 0.01 M citrate buff er (pH 6.0), or sodium metaperiodate, The treatments were applied sepa rately and combined, After labeling, crystalloids in untreated section s had a probe density of 5 particles/mu m(2). Crystalloids in sections treated only with sodium ethoxide or sodium metaperiodate had probe d ensities of 15-20 particles/mu m(2). Sodium ethoxide combined with hea ting on water, or citrate buffer, gave probe densities of 140-160 part icles/mu m(2). Sodium metaperiodate combined with heating on citrate b uffer gave the highest probe density (195 particles/mu m(2)). Although sodium ethoxide coupled with heating increased probe density, the eth oxide etched the sections and caused unacceptable damage, Treatment wi th sodium metaperiodate followed by heating on citrate buffer is recom mended for antigen unmasking, This combination gave a high probe densi ty and sections remained intact, with good ultrastructural detail.