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.
D000818:Animals; D001923:Brain Chemistry; D002795:Choline O-Acetyltransferase / Q000276:immunology*; D002799:Cholinergic Fibers / Q000032:analysis; D003035:Cobalt; D006651:Histocytochemistry; D007124:Immunoenzyme Techniques; D037102:Lectins / Q000276:immunology; D051381:Rats; D011919:Rats, Inbred Strains; D014909:Wheat Germ Agglutinins
D037102:Lectins; D014909:Wheat Germ Agglutinins; D003035:Cobalt; D002795:Choline O-Acetyltransferase