Interactions of immunostained ACTH1-39 fibers and CRF neurons in the paraventricular nucleus of rat hypothalamus: application of avidin-glucose oxidase to dual immunostaining procedures.

Dual staining immunocytochemical procedures were used to elucidate the distribution and potential anatomic relationship of corticotropin releasing factor (CRF)-containing cell bodies and central opiocortin fibers in the paraventricular nucleus (PVN) of rat hypothalamus. Double-immunostained preparations employed antibodies and the peroxidase-antiperoxidase (PAP) technique as a first sequence in the immunostaining protocol, followed by antibodies and glucose oxidase-conjugated avidin as the second sequence, yielding rich brown and vibrant blue reaction products respectively. Distinctive features of this new dual immunostaining technique and its applicability to the study of co-localization and coexistence of neuropeptides are discussed. This study demonstrates immunostained ACTH1-39 fibers in intimate anatomic proximity to, and often surrounding in remarkable density, CRF-containing cells localized in distinct subnuclei of the PVN. It appears that the central opiocortin system selectively innervates CRF neurons in the PVN.