Retrograde horseradish peroxidase tracing combined with localization of choline acetyltransferase immunoreactivity

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.