Retrograde horseradish peroxidase tracing combined with localization of choline acetyltransferase immunoreactivity.

Abstract

Localization of choline acetyltransferase (ChAT) immunoreactivity in rodent brain (AI Levey, DM Armstrong, SF Atweh, RD Terry, BH Wainer: J Neurosci 3 1, 1983) with a monoclonal antibody (Ab8) has been previously reported. Now a procedure for combining ChAT immunohistochemistry with retrograde tracing for the purpose of mapping cholinergic pathways is presented. Rats were injected with horseradish peroxidase-wheat germ agglutinin in the tongue and cerebral cortex. Sections from their perfusion-fixed brains were reacted with 3,3'-diaminobenzidine (DAB)/H2O2/cobalt acetate followed by ChAT localization with monoclonal antibody Ab8 using the peroxidase-antiperoxidase method and visualization using DAB/H2O2. Double-labeled cells were visualized with black punctate staining (retrograde tracer) on a diffuse brown cytoplasmic background (ChAT immunoreactivity) in the hypoglossal nucleus and ventral telencephalon (substantia innominata-nucleus basalis). Conditions of fixation, histochemistry, and immunohistochemistry that contribute to optimal resolution for this procedure are discussed.