Pyrocatechol as a stabilizing agent for o-tolidine and o-dianisidine: a sensitive new method for HRP neurohistochemistry.

A new procedure for detecting HRP in nerve tissue is described which is based on the use of pyrocatechol to stabilize the oxidation products of o-tolidine and o-dianisidine in citric acid/ammonium acetate buffer of pH 4.85. In both cases the precipitate obtained is insoluble, stable and more visible than when any variant of the diaminobenzidine method is employed, and the morphological image of neurons and nerve fibres labelled with HRP is superior to that produced by the tetramethylbenzidine/sodium nitroprusside method. There is no non-specific precipitation, and no retraction of nerve tissue has been observed. The performance of the method is improved further using either o-tolidine/pyrocatechol or o-dianisidine/pyrocatechol in conjunction with glucose oxidase, which may be useful if it is desired to obtain Golgi-like images of HRP-bearing cells or to display weakly HRP-labelled nerve fibres.