A new method for the cytochemical demonstration of peroxidase for light, fluorescence and electron microscopy

A new technique for the cytochemical demonstration of peroxidase is presented. Monomeric homovanillic acid is converted by H2O2 and peroxidase into its dimeric form, which is then precipitated as a complex salt of lead and rhodamine 6G or rhodamine B. The reaction product can be visualized by conversion to lead sulphide or viewed directly under the fluorescence microscope, since it emits a red fluorescence when excited with green light. The reaction is rapid and results in good localization at the cytologic level of peroxidase activity in granulocytes. The technique can be applied for the ultrastructural localization of enzymatic activity, but in its present form it does not match the localization sharpness of the diaminobenzidine method. This fluorescent cytochemical technique will also detect horseradish peroxidase activity and may provide a usefull probe in peroxidase immunohistochemistry. The principle of complexing metal salts with fluorescent dyes may find a more general application in enzyme cytochemistry.