A comparison of eight different chromogen protocols for the demonstration of immunoreactive neurofilaments or glial filaments in rat cerebellum using the peroxidase-antiperoxidase method and monoclonal antibodies.

Eight different previously described chromogen protocols were evaluated with respect to their sensitivity for the visualization of horseradish peroxidase (HRP) in a peroxidase-antiperoxidase (PAP) complex used with the unlabeled antibody method for immunohistochemistry. The protocols were evaluated in a test system that involved the demonstration of immunoreactive neurofilaments (NF) or glial filaments (GF) in paraffin-embedded sections of rat cerebellum using anti-NF or anti-GF monoclonal antibodies (MA). The chromogens included: amino-ethylcarbazole (AEC), diaminobenzidine (DAB), O-tolidine, paraphenylenediamine-pyrocatechol (PPD-PC), and tetramethylbenzidine (TMB). The incubation medium using DAB as the chromogen was employed at neutral pH, at pH 5.1, or at neutral pH with the addition of either cobalt chloride or imidazole to intensify the reaction product. The relative sensitivity of the chromogen protocols was quantitated by comparing the dilution of the anti-NF or anti-GF MA at which NF or GF immunoreactivity was extinguished using each protocol. The results obtained with both the anti-NF and anti-GF MA indicated that DAB with imidazole was the most sensitive chromogen protocol.