NADH-Dehydrogenase Reaction in Combination with Immunoperoxidase (PAP) Staining for Light Microscopic Observation on the Interneuronal Relations of the Enteric Nervous System of the Pig

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Abstract
A novel procedure for a simultaneous demonstration of particular enteric nerve cell types and peptidergic nerve fibres has been developed by combining the histochemical reaction for NADH-dependent dehydrogenase and the unlabelled antibody peroxidase-antiperoxidase (PAP) method described by Sternberger. Whole-mount spreads were successively incubated in a NADH: nitroblue tetrazolium solution, fixed with a picric acid/ formaldehyde mixture, dehydrated, cleared and rehydrated before processing for immunocytochemical localization of the neuropeptide by the PAP method. The nerve cells appear heavily stained by deposits of dark blue formazan, whereas the peptide-containing nerves appear bright brown. In the myenteric and submucous plexuses of the porcine small intestine the devised method allows an appropriate identification of Dogiel’s type I, type II and type III neurons surrounded by varicose enkephalin-like immunoreactive fibre baskets with button-like twigs to the very surface of the ganglionic cells, suggestive of synaptic connections.