Fixation, fine structure, and immunostaining for neuropeptides: perfusion versus immersion of the neuroendocrine hypothalamus

The effects of various fixatives and fixation methods on ultrastructural morphology and the immunocytochemical localization of beta-endorphin were examined in rat brain. The mediobasal hypothalamus was preserved by vascular perfusion and/or immersion in nine different fixatives. We tested several combinations of paraformaldehyde, glutaraldehyde, acrolein, and picric acid in various isosmolar buffers. Vibratome sections were stained for beta-endorphin employing the peroxidase-antiperoxidase technique, or processed directly for electron microscopy. The ultrastructural quality of a given region was attributed to its location with respect to the blood-brain barrier, the method of fixation, and the concentrations of some of the fixative components. Immersion fixation gave better results and reduced extracellular space in the median eminence (outside the blood-brain barrier) and areas close to the hypothalamic surface. Positive immunostaining of beta-endorphin perikarya occurred only in tissue fixed with periodate-lysine-paraformaldehyde. Light to moderate fiber staining was also present in some paraformaldehyde-glutaraldehyde-acrolein combinations. However, a glutaraldehyde concentration of 1% or higher abolished all positive staining for beta-endorphin. These results emphasize the necessity of optimizing fixation for ultrastructure and for immunocytochemical staining of each individual antigen. The choice of the best fixation method depends not only on the intracellular location of the antigen but also on the relationship between hypothalamic tissue compartments and the blood-brain barrier.