A new stabilizing agent for the tetramethyl benzidine (TMB) reaction product in the histochemical detection of horseradish peroxidase (HRP).

In this paper, an alternative procedure for the histochemical detection of HRP using amonium heptamolybdate (AHM) as a stabilizing agent and tetramethyl benzidine (TMB) as a chromogen is reported. This procedure avoids the two main problems that occur in previous methods using sodium nitroferricyanide (SNF) as the stabilizer, namely, the appearance of needle-shaped crystals at non-specific anatomical sites, and intensive tissue shrinkage. A comparative study of both, the TMB-AHM and TMB-SNF methods, was performed in the analysis of cerebral cortex afferent connections of the lizard Podarcis hispanica. This study demonstrates that the two methods are of similar sensitivity. The TMB-AHM reaction can be carried out at physiological pH (from 6 to 8), thus, avoiding tissue contraction. The reaction product is of an intense blue-green colour and, as with the TMB-SNF method, shows granulation. The appearance of non-specific precipitates is completely avoided when the incubation medium is maintained at a pH in excess of 5.