9] Localization of hormones with the peroxidase-labeled antibody method

Abstract

Abstract Efficient conjugation of peroxidase to anti-rabbit immunoglobulin can be achieved through the carbohydrate moiety of peroxidase without significant loss of either enzymatic or antibody activities. Such conjugates are suitable for the localization of specific antigens at both the light and electron microscopic levels. When Bouin's or Zamboni's fixatives were used, no problem in retaining hormone antigenicity was encountered. At the light microscopic level, since different colored substrates for peroxidase are available, it is possible to localize more than one antigen on the same tissue section. At the electron microscopic level, the choice of embedding medium is an important factor affecting the preservation of antigenicity. Although Epon and methacrylate are useful for all hormones if the histochemical reaction is done prior to embedding and for some hormones if the reaction is done after embedding, we have had more generalized success with polyethylene glycol. Furthermore, the use of polyethylene glycol permits the same antigen in the same tissue section to be localized by both light and electron microscopy. Using specific rabbit antisera, a suitable peroxidase substrate, and peroxidase conjugated to anti-rabbit immunoglobulin, each of the various pituitary and pancreatic hormones has been localized. We therefore regard the peroxidase-labeled antibody method as a very powerful immunohistochemical procedure for the light and electron microscopic localization of hormones.