We examined the suitability of the freeze-substitution and paraffin embedding technique for the im-munolocalization of membrane-bound and soluble antigens in the rat at the light microscopic level. Frozen tissues were substituted at -80°C in methanol or acetone containg chemical fixatives, i. e., formaldehyde, glutaraldehyde, acrolein, hexamethylene diisocyanate (HMD), diethyl malonimide (DEM) and 1-ethyl-3- (3-dimethyl-aminopropyl) carbodiimide (water soluble carbodiimide, WSC). The following antigens were localized using monoclonal antibodies and antisera: A) membrane-bound antigens; γ-glutamyl transpeptidase (γ-GTP), common antigen of secretory granule membrane (SG 170 antigen) and Golgi associated antigen (GAA 108); and B) soluble antigens; branched-chain amino acid transferase type I isozyme (BAT), glutamate dehydrogenase (GDH), pancreatic amylase, proliferating cell nuclear antigen (PCNA), rat serum albumin and IgG.
1) The freeze-substitution technique maintained an excellent tissue structure and conservation of antigenicity. By using this method, BAT was localized in mitochondria in iver cells, and γ-GTP was demonstrated in the secretory granule membrane of pancreatic acinar cells, although conventional fixation methods provided negative reaction. 2) In general, the membrane-bound antigens were localized in detail with a strong immunoreaction in the tissues substituted in solvent alone; however, for the localization of soluble antigens, tissues substituted in solvents containing chemical fixatives revealed a strong and precise antigen localization. 3) Formaldehyde and glutaraldehyde proved to be better fixatives concerning the conservation of structure and antigenicity than the other chemical reagents. 4) The choice of substitution solvent was important for the immunohistochemistry of some antigens. For example, methanol was suitable for PCNA, and substitution in acetone was essential for γ-GTP using one of the monoclonal antibodies to γ-GTP.