Immunohistochemical localization of antigens in tissue embedded in JB-4 by backscattered electron imaging utilizing colloidal gold-labeled and peroxidase-labeled antibodies.

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Backscattered electron image (BEI) of colloidal gold-labeled antibody was compared with that of osmium which has chelated with the reaction product of peroxidase (HRP)-labeled antibody. Male rat hypophyses were fixed with 4% formaldehyde in 0.1M phosphate buffer, pH 7.4 and embedded in JB-4. Tissue sections at 2μm in thickness were placed on carbon coated glass slides and were reacted with rabbit anti-ACTH followed with either colloidal gold-labeled or HRP-labeled goat anti-rabbit IgG. Those reacted with HRP-labeled antibody were incubated with a solution containing diaminobenzidine (DAB) and H2O2. The DAB reaction product was chelated then with OsO4→thiocar-bohydrazide→OsO4. After washing and drying, the sections were observed with BEI mode of a JEOL T200 scanning electron microscope at 25kV accelerating voltage. BEI from the colloidal gold-labeled antibody was visualized as very fine granules in the cytoplasm of ACTH cells at high magnification (>3, 000×), but was difficult to recognize at low magnification (<300×). On the other hand, BEI from the osmium chelated with the reaction product of HRP could be recognized as amorphous masses in the cells both at the low and high magnifications. From these observations, it was concluded that the concomitant use of colloidal goldlabeled antibody and HRP-labeled antibody should be useful for the immunohistochemical localization of more than one antigen on the same section mounted on an ordinary glass slide at the electron microscopic level.

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