The unlabeled antibody method. Contrasting color staining of beta-lipotropin and ACTH-associated hypothalamic peptides without antibody removal.

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Abstract
Staining patterns of 3 antisera to ACTH peptides and antiserum to ovine .beta.-lipotropin were compared to reveal degrees of nonidentity of hypothalamic immunoreactivity with that found in the pituitary [in rats]. Dual color immunocytochemistry was used to reveal association of these reactivities with those of other hypothalamic peptides. Cells of the pars intermedia were stained with each of the 4 antisera. Fibers in the dorsomedial nucleus and cell bodies and fibers in the arcuate nucleus and preoptic area were stained with anti-ACTH1-39 and 1-24, but remained unstained with anti-ACTH17-39 and anti-.beta.-LPH. In the supraoptic and paraventricular nuclei and in the internal zone of the median eminence of all rats and in the pars nervosa of occasional rats, reactivity for anti-.beta.-LHP and anti-ACTH1-39 were found in the same perikarya and fibers as vasopressin. Anti-ACTH1-24 and 17-39 failed to react in the magnocellular system. The hypothalamic reactivities of anti-ACTH1-24 and anti-.beta.-LPH appeared to be specific for peptides resembling the immunizing antigens. Reactivity of anti-ACTH1-39 was due to ACTH-like material in the dorsomedial-arcuate area, but to neurophysin II in the magnocellular system. ACTH reactivity in the dorsomedial-arcuate area resembles that of pituitary ACTH; ACTH reactivity in the magnocellular system reflects an antigen unrelated to pituitary ACTH or at best possessing only partial homology with it. .beta.-LHP and ACTH are derived from different precursors in the hypothalamus and pituitary, or alternatively, cleaved from a common precursor by different enzymes. The findings support the concept of regional diversity of neuropeptides and of neuronal idiotypy.