Hypothalamus and pituitary tissues from monkey and calf, fixed in Bouin’s or formalin and embedded in paraffin, were deparaffinized and rehydrated and examined for the localization of neurophysin using immunoenzyme techniques. Rabbit antiserum to bovine neurophysin I was used as the specific antibody, with peroxidase labeled antirabbit globulin in the 2-layer method, and with unlabeled antirabbit and rabbit antiperoxidase sera followed by peroxidase in the 3-layer immunobridge technique. The brown reaction products of the localized peroxidase with 3, 3’ diaminobenzidine were seen by light microscopy. Tissues were counterstained by cresyl violet or aldehyde fuchsin. Neurophysin I levels in pooled cryostat sections of bovine supraoptic (SO) and paraventricular (PV) hypothalamic regions were determined by radioimmunoassay. With these methods, in both species neurophysin is localized in (1) SO and PV neuronal perikarya, (2) in their axons coursing through hypothalamus and (3) in their terminals in posterior pituitary. Neurons scattered along the PV tract and Herring bodies of the posterior pituitary also contain neurophysin. No neurophysin is found in the posterior pituitary glial cells and in other areas of the central nervous system. Counterstaining with cresyl violet indicated that a significant number of SO and PV neurons do not contain neurophysin material. Sections counterstained with aldehyde fuchsin suggest that neurophysin might be an integral part of the neurosecretory material. The specificity of the antibody for neurophysin I, when tested on bovine tissues using the antiserum differentially absorbed with neurophysin I or neurophysin II, showed that neurophysin I is localized in both SO and PV neurons. Tissue levels of neurophysin I in extracts of bovine SO and PV regions by radioimmunoassay confirm these findings.
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