A novel tetrazolium method for peroxidase histochemistry and immunohistochemistry

We developed a new method for the histochemical demonstration of peroxidase. This method, which has a novel reaction mechanism, is based on the oxidation of phenol by peroxidase and coupling of this reaction to the reduction of a tetrazolium salt, with the deposition of an insoluble formazan at sites of enzyme activity. This new method was compared with an established diaminobenzidine (DAB) technique for peroxidase histochemistry and immunohistochemistry. Although both methods identified peroxidase activity in myeloid cells of bone marrow biopsy specimens, there was no interference from red cell pseudoperoxidase activity with the phenol-tetrazolium method, in contrast to the diaminobenzidine method. The detection of cytokeratin using an indirect immunoperoxidase technique was compared with both methods for demonstrating peroxidase activity. The phenol-tetrazolium method gave results similar to that obtained with DAB and appeared to be at least as sensitive as DAB in detecting low amounts of antigen. In addition, the production of a formazan as the final reaction product means that the phenol-tetrazolium method is ideally suited for quantitative peroxidase histochemistry. Therefore, the phenol-tetrazolium method represents a useful alternative method to DAB and for certain applications offers significant advantages over DAB.