Simultaneous localization of multiple tissue antigens using the peroxidase-labeled antibody method: a study on pituitary glands of the rat.

TLDR: The peroxidase-labeled antibody method was modified to localize multiple tissue antigens in a single histologic section, using substrates that develop reaction products of different colors to identify the antigenic sites.

Abstract: The peroxidase-labeled antibody method was modified to localize multiple tissue antigens in a single histologic section. The first antigen was localized by the indirect method and the antisera were removed from the section by elution, leaving the colored reaction products identifying the antigenic sites. The second and subsequent antigens were localized similarly, using substrates that develop reaction products of different colors. For the demonstration of the method, anti-human growth hormone (GH), anti-human thyrotropic hormone (TSH) and anti-human chorionic gonadotropin, which cross-reacts with rat luteinizing hormone (LH), were used with sections of pituitary gland of the rat. AntiGH reacted with small acidophilic cells. Anti-LH was localized in oval shaped cells, and anti-TSH was localized in angulated basophilic cells. Some cells were devoid of these three antigens and no cell contained more than one of them. A convenient method which would identify more than one amitigen in the same section of tissue would be of considerable value to cellular biologists. In the past, two tissue antigens have been localized simultaneously with the immunofluorescent method using antibodies labeled with fluorescent coml)ounds of different color (4, 5, 1012). This technique has obvious limitations, including the tendency of the fluorescence of one label to mask that of the other, and the tendency for one color to fade more rapidly than the other