title: Stabilization of the tetramethylbenzidine (TMB) reaction product: application for retrograde and anterograde tracing, and combination with immunohistochemistry.
abstract: Tetramethylbenzidine (TMB) as a substrate for horseradish peroxidase (HRP) histochemistry is more sensitive than other chromogens. Its instability in aqueous solutions and ethanol, however, has limited its application. We now report a method for stabilizing TMB by incubation in combinations of diaminobenzidine (DAB)/cobalt (Co2+)/H2O2. The stabilized TMB product was unaffected by long-term exposures to ethanol, neutral buffers, and subsequent immunohistochemical staining procedures. A procedure is recommended for optimal stabilization of TMB that affords a sensitivity for demonstrating retrogradely labeled perikarya comparable to standard TMB histochemistry. The physical characteristics of the reaction product make it suitable for combination with the unlabeled antibody, peroxidase-antiperoxidase (PAP) immunohistochemical staining procedure. This was established by staining retrogradely labeled neurons in the basal forebrain with a monoclonal antibody against choline acetyltransferase. Because the stabilized TMB product exhibited a superior sensitivity over cobalt ion intensification of the DAB-based reaction product (DAB-Co), it offers a distinct advantage over previously described combination procedures.
mesh_terms: D000818:Animals; D001560:Benzidines; D002540:Cerebral Cortex / Q000378:metabolism; D004355:Drug Stability; D006651:Histocytochemistry / Q000379:methods*; D006735:Horseradish Peroxidase; D007120:Immunochemistry / Q000379:methods; D037102:Lectins; D008297:Male; D051381:Rats; D011919:Rats, Inbred Strains; D013696:Temperature; D014909:Wheat Germ Agglutinins
chemical_list: D001560:Benzidines; D037102:Lectins; D014909:Wheat Germ Agglutinins; C021758:3,3',5,5'-tetramethylbenzidine; D006735:Horseradish Peroxidase