Immunohistochemical localization of acid alpha-glucosidase in rat liver

Acid alpha-glucosidase (E.C. 3.2.1.3) was purified more than 60,000-fold from rat liver. Antibody was obtained by injection of this pure enzyme into rabbits with Freund's complete adjuvant. The resultant anti-acid alpha-glucosidase immunoglobulin (Ig) G was digested with pepsin and then F(ab')2 was treated with 2-mercaptoethanol. Coupling of Fab' to horseradish peroxidase was performed according to the method of Wilson and Nakane. Light microscopic observation of the immunohistochemical localization of this enzyme in rat hepatocytes revealed small granular deposits of diaminobenzidine reaction products. The reaction diffusely observed in the hepatocyte cytoplasm of any area. Under the electron microscope, the reaction precipitates were found to be located on the lysosome membrane, particularly on the inner side of the membrane, as small dots. The small vesicles were strongly positive for this reaction. Occasionally positive reaction were also demonstrated in the lumen of the secondary lysosomes. However, the Golgi and its associated structures did not show a positive reaction.