Light and electron microscopic immunocytochemistry of the same nerves from whole mount preparations

Abstract

A technique for performing correlated light and electron microscopic immunocytochemical studies on whole mount preparations has been developed using myenteric plexus from guinea pig small intestine as a model. With this method a structure containing a particular antigen can first be located by light microscopy and then examined with the electron microscope. Pieces of intestine pinned on balsa were incubated in oxygenated Krebs solution at 37° C for 90-120 min and then fixed for 1 hr at room temperature in 4% formaldehyde, 0.05% glutaraldehyde, and 0.2% picric acid in 0.1 M sodium phosphate buffer, pH 7.4. The tissue was washed vigorously in several changes of 50% ethanol until the picric acid had been removed, stored overnight in phosphate buffer, and then exposed to 0.1% sodium cyanoborohydride in buffer for 30 min. Vasoactive intestinal peptide (VIP) was localized in separated layers containing myenteric plexus and longitudinal muscle using the peroxidase-antiperoxidase technique with imidazole intensification of the diaminobenzidine reaction product. At the light microscope level, tissue stained by this technique showed VIP-immunoreactive nerve cell bodies and process throughout the thickness of the myenteric ganglia in numbers approximately equivalent to those seen in whole mounts processed by an established technique for the light microscopic demonstration of VIP, which does not involve exposure of tissue to glutaraldehyde. VIP-immunoreactive structures that were first identified at the light microscope level were subsequently examined at the electron microscope level. VIP-immunoreactive axon profiles were found to form synapses on both immunoreactive and nonimmunoreactive myenteric neurons. The fine structural appearance of the different cell types present in whole mount preparations prepared by this method was similar to that seen in conventionally fixed tissue, except that free and bound ribosomes were absent from the tissue processed for immunocytochemistry. The method described here is reliable and no more difficult than presently available methods for preembedding electron microscopic immunocytochemistry on sections. Its main advantage is that immunoreactive structures for ultrastructural study can be selected from the entire population of chemically identified nerves within a whole mount rather than from a smaller sample present within a section. This technique is applicable to other tissues that can be stained immunohistochemically in whole mounts. The fixation and penetration enhancement procedures can also be adapted for immunocytochemical studies on vibratome or frozen sections.