Immunocytochemical detection of peptides in osmicated and plastic-embedded tissue. An electron microscopic study

In an attempt to improve the ultrastructural preservation of tissue immunostained with the postembedding staining technique, we verified the effect of postfixation with OsO4 on the antigenicity of several pituitary hormones and neuropeptides. For this purpose, the rat pituitary and central nervous system (CNS) were perfused with 2.5% glutaraldehyde and postfixed in two different ways: a) postfixation by immersion of small fragments in a mixture of 1% OsO4 and 1% potassium ferrocyanide, and b) postfixation with perfusion of 500 ml of 0.5% OsO4. The results obtained were similar with the two types of postfixation. In the pituitary gland, all the hormones could be very easily detected, although the staining was less intense for the glycoprotidic hormones. In the CNS the following neuropeptides: somatostatin, luteinizing hormone-releasing hormone (LHRH), Leu-enkephalin and substance P could be immunostained. These results indicate that a variety of polypeptidic antigens, including small neuropeptides, can survive postfixation with OsO4. In both the pituitary and CNS, the ultrastructural preservation was very good with a high contrast of membranes, thus permitting a clear identification of positive organelles. Moreover, it appears that, since the synaptic junctions are well-preserved after postfixation with OsO4, postembedding staining can now be utilized to identify and classify the different categories of endings containing neuropeptides.