The unlabeled antibody method. Contrasting color staining of paired pituitary hormones without antibody removal.

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Abstract
Two antigens were localized with the unlabeled antibody peroxidase-antiperoxidase (PAP) technique by carrying out the immunocytochemical reaction in 2 sequences. In the 1st sequence, antiserum to the 1st antigen was followed by anti-immunoglobulin, PAP, H2O2 and diaminobenzidine tetrahydrochloride (DAB) to yield a brown reaction product. In the 2nd sequence, antiserum to the 2nd antigen was again followed by anti-immunoglobulin and PAP, and then by H2O2 and 4-chloro-1-napthol (CN) to yield a blue reaction product. Even though the same anti-immunoglobulin and PAP were used in both sequences, and even though H2O2 was applied as enzyme substrate twice, no color mixing occurred. It was unnecessary to remove the immunoreagents of the 1st staining sequence prior to applying the 2nd sequence. Apparently, the DAB reaction product masked antigen and catalytic sites of the 1st sequence of immunoreagents and thus prevented interaction with reagents of the 2nd sequence. This conclusion was derived from attempts at staining the same antigen with the same primary antiserum in the 1st and 2nd sequence. Only brown reaction product was obtained and no color mixing occurred. When either primary antiserum or DAB in the otherwise complete 1st reaction sequence was progressively diluted, colors became mixed until, upon omission of primary antiserum or DAB, standard (control) blue was obtained. Similarly, standard brown was obtained when primary antiserum was omitted in the 2nd sequence. In the pars distalis of the [rat] pituitary, separate cells stained brown and blue when pairs of antisera to the following hormones were applied: growth hormone, prolactin and either ACTH1-24 or ovine .beta.-lipotropin. Separate cells were also visualized when anti-ACTH1-24 was followed by anti-LH [lutropin]. When anti-growth hormone was followed by anti-LH, a few cells were mixed in color, even though most cells were either brown or blue. When anti-ACTH1-24 was followed by anti-ACTH1-39, mixed color staining occurred in cells of the pars intermedia and distalis, and fibers in the pars nervosa were blue.