Abstract
A procedure has been developed for the cytochemical localization of acetylcholine (ACh) receptors by means of the snake neurotoxin α-bungarotoxin (α-Btx) labeled directly with horseradish peroxidase (HRP). Conjugation of HRP to α-Btx was carried out according to the method of Nakane and Kawaoi (1). A fraction of the conjugate with a MW of ∿48,000 was separated from free peroxidase and heavier MW fractions by column chromatography. This fraction corresponds to a conjugate in which one molecule of HRP is coupled to one of α-Btx. It was found to retain physiological activity since it produced a postsynaptic blockade at frog sartorius neuromuscular junction similar to that obtained with native α-Btx. For localization of receptors, mouse diaphragm, frog sartorius, or newt limb muscles were incubated in the conjugate for from one half to two hours, fixed in glutaraldehyde, reacted with H2O2 and diaminobenzidine for peroxidase activity, fixed in osmium tetroxide, and processed for electron microscopy.
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