Quantitative Immunocytochemistry of Corticotropin by the Unlabeled Antibody Enzyme Method with and without Peroxidase-Antiperoxidase Complex (PAP)

The unlabeled antibody enzyme method localizes antigen by reaction with (1) specific antiserum (primary antibodies), (2) antiserum to the primary immunoglobulin, (3) antiperoxidase (anti-PO) and (4) peroxidase (P0). The reaction is developed with hydrogen peroxide as substrate for P0 and diaminobenzidine (DAB) as co-substrate, followed by osmication. Anti-PO can be used in the form of antiserum (procedure A), purified antibody (procedure B), or steps (3) and (4) can be combined by the use of purified, soluble PAP (procedure C). Only at relatively low resolutions does identification of antigen depend on specific staining. High resolution localization depends on identification of the characteristic ring-shaped PAP molecules (Fig. 1).We have compared the sensitivities of procedures A, B and C upon staining serial Araldite sections of rat pituitary intermediate lobes using anti17-39 corticotropin as primary antiserum. PAP contained 3.33 mg anti-PO, 1.2 mg PO/ml and was essentially pure. It was employed at a standard dilution of 1:50. Antiserum and purified antibody to PO were used at standard dilutions containing equivalent amounts of anti-PO (1:24 for antiserum, 1:5 for purified antibody).