Differential sensitivity between blue and brown reaction procedures for HRP neurohistochemistry

Four rhesus monkeys and two rats were injected with horseradish peroxidase (HRP). Adjacent sections were stained with a 3,3'-diaminobenzidine tetrahydrochloride (DAB) and a benzidine dihydrochloride (BDHC) procedure which yields a blue reaction product at sites of HRP activity. The number of labeled neurons was counted within the same regions of the adjacent sections with bright-field and dark-field illumination. Sections processed with the BDHC procedure consistently contained more labeled neurons than adjacent sections processed with a representative DAB procedure. Such differences were observed in cortical as well as subcortical areas and in the monkey as well as in the rat. Moreover, the visibility of labeled neurons under bright-field illumination was quite superior in the tissue stained with the BDHC procedure. It is concluded that the BDHC procedure is more sensitive than a DAB procedure for the demonstration of the retrograde transport of HRP. In additional experiments tetramethyl benzidine (TMB) was used in place of BDHC and comparable results were obtained.