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

Abstract

Two antigens were localized with the unlabeled antibody peroxidase-antiperoxidase (PAP) technique by carrying out the immunocytochemical reaction in two sequences. In the first sequence, antiserum to the first antigen was followed by anti-imunoglobulin, PAP, hydrogen peroxide and diaminobenzidine tetrahydrochloride (DAB) to yield a brown reaction product. In the second sequence, antiserum to the second antigen was again followed by anti-immunoglobulin and PAP, and then by hydrogen peroxide 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 hydrogen peroxide was applied as enzyme substrate twice, no color mixing occurred. Therefore, it has been found unnecessary to remove the immunoreagents of the first staining sequence prior to applying the second sequence. Apparently, the DAB reaction product masked antigen and catalytic sites of the first sequence of immunoreagents and thus prevented interaction with reagents of the second sequence. This conclusion had been derived from attempts at staining the same antigen with the same primary antiserum in the first and second sequence. Only brown reaction product was obtained and no color mixing occurred. However, when either primary antiserum or DAB in the otherwise complete first reaction sequence were 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 second sequence. In the pars distalis of the 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 β-lipotropin. Separate cells were also visualized when anti-ACTH1-24 was followed by anti-LH. 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.