Improvement of the tetramethyl benzidine reaction with ammonium molybdate as a stabilizer for light and electron microscopic ligand-HRP neurohistochemistry, immunocytochemistry and double-labelling

Ammonium heptamolybdate (AHM) was used as a stabilizing agent in the tetramethyl benzidine (TMB) reaction of choleragen subunit B conjugated horseradish peroxidase (CB-HRP) neurohistochemistry (TMB-AHM method). In comparison with Mesulam's TMB method employing sodium nitroprusside as a stabilizing agent (TMB-SNP method), the TMB-AHM procedure offfers a similar sensitivity with regard to the visualization of CB-HRP labelled neurons and their extranuclear Golgi-phobic dendrites. However, it is less sensitive for the demonstration of anterogradely transported CB-HRP in axon terminals. At the nearly physiological pH value of the reaction medium (pH 6–8), it demonstrates better preservation of tissue and cell structures in the reacted sections. Under the electron microscope, the specific reaction product can be clearly distinguished and little damage of cellular and subcellular structures occurred. Preliminary application of TMB-AHM method to choleragen subunit B (CB) immunocytochemistry and double labelling technique which paired the neuronal tracing methods of HRP neurohistochemistry and CB immunocytochemistry, was also carried out with small modification.