A double immunohistochemical labeling technique was developed with two immunoperoxidase reactions performed before and after embedding in epoxy resin using two different chromogens: diaminobenzidine (yellowish brown color) and diaminobenzidine + nickel ammonium sulfate (black color). The two catecholamine enzyme immunoreactivities (phenylethanolamine-N-methyltransferase, and tyrosine-hydroxylase) were not found in the same cells, while gamma-aminobutyric acid (GABA) immunoreactivity was observed in large tyrosine-hydroxylase immunoreactive cells, but not in phenylethanolamine-N-methyltransferase immunoreactive cells.
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