Simultaneous double immunoenzymatic labeling: a new procedure for the histopathologic routine.

We developed a new procedure for simultaneous immunohistoenzymatic double labeling that uses two mouse monoclonal antibodies of different isotypes or two antibodies from different species. The technique is a combination of the alkaline phosphatase-antialkaline phosphatase, avidin-biotin, and digoxigenin methods and uses horseradish peroxidase and alkaline-phosphatase detection systems. This standardized procedure can be carried out easily in the routine of a pathology laboratory and performed in parallel with single-antigen immunostaining, without the need for extra material or additional time for the laboratory technicians. This method can be applied to the detection of antigens localized in distinct cells or subcellular compartments and to the covisualization of different components from the same subcellular compartment.