Simultaneous visualization by light microscopy of two pituitary hormones in a single tissue section using a combination of indirect immunohistochemical methods.

Two indirect methods involving enzyme-labeled antibodies were used to demonstrate simultaneously two distinct tissue antigens in the same histologic section without a need for antigen-antibody dissociative procedures. Sections of rat pituitary gland were incubated with rabbit anti-rat luteinizing hormone followed by goat anti-rabbit gamma-globulin conjugated to horseradish peroxidase. The same sections were then further incubated with monkey anti-rat growth hormone followed by goat anti-monkey gamma-globulin conjugated to glucose oxidase. Antigenic luteinizing hormone was subsequently localized with hydrogen peroxide-3,3'-diaminobenzidine as substrate for peroxidase, and growth hormone was localized with a glucose-phenazine methosulfate-nitroblue tetrazolium mixture as a substrate for glucose oxidase. The method relies on the availability of specific primary antibodies raised in different animal species in addition to corresponding specific secondary antibodies linked covalently to separate enzymes.