Effect of fixation on the demonstration of neurophysin and "Gomori-positive" substances in neurosecretory granules of the rat hypothalamus.

Proteins reacting with neurophysin antibodies and "Gomori-positive" substances were demonstrated histochemically in hypothalamic neurosecretory material of normal and bilaterally adrenalectomized rats after two different fixations: a) picric acid-formalin (PAF) for 7 days at 37 degrees C; b) Bouin's fluid for 20 h at 4 degrees C. After PAF-fixation anti-neurophysin reactive neurosecretory granules are found in all parts of the supraoptico-hypophysial system and in the suprachiasmatic nucleus of normal and adrenalectomized animals. In the latter they can additionally be demonstrated in the outer layer of the median eminence. Amount and distribution of "Gomori-positive" substances correspond to those described for the immunoreactive material, except for the suprachiasmatic nucleus, in which the substances can not be detected; Following fixation in Bouin's fluid the immunohistochemical reactions are unchanged whereas the staining of "Gomori-positive" substances is remarkably impaired. The amounts of the substances demonstrable in the neural lobe are diminished and in the cells of the supraoptic and paraventricular nuclei as well as in both median eminence layers only traces of the substances are to be seen. The findings indicate that negative results in demonstrating "Gomori-positive" substances may be caused by inappropriate fixation and need to be controlled by immunohistochemical techniques.