Time of fixation and the localization of gomori-positive and neurophysin-containing structures in the rat hypothalamus

Abstract

Rats were decapitated and the complete head stored at 4°C. At various time intervals, up to a maximum of 14 days, the brains and pituitary glands were removed and fixed in formalin-picric acid. Neurosecretory material (NSM), as revealed with crotonaldehyde fuchsin, and neurophysin as demonstrated by means of immunoperoxidase histochemistry were localized in the hypothalamic magnocellular nuclei, median eminence and posterior pituitary gland. NSM and neurophysin were present in the paraventricular (PVN) and supraoptic (SON) nuclei up to periods of 14 days although the cellular morphology was disrupted after about 4 days. After 4 days there was an abundance of positive staining “droplets” in both the SON, PVN and along the nerve fibres of the supraoptico-neurohypophysial tract. Pituitary gland NSM and neurophysin were still present 14 days post mortem. The slab gradient polyacrylamide gel electrophoretic pattern of soluble proteins extracted from the posterior pituitary gland from the experimental and normal animals showed almost complete identity confirming the minimal action of proteolytic enzymes at 4°C up to a period of 6 days. It is concluded that as long as the tissue is cooled, rapid fixation of hypothalamic tissue is not essential for the localization of NSM and neurophysin.