The use of metal-catalyzed oxidation to suppress background staining caused by marker amplification reagents and the effects of this oxidation on the stability of antibody-antigen complexes in immunohistochemistry

Marker amplification is a powerful technique for visualizing immunohistochemically deposited markers that otherwise would be invisible. Amplification usually is achieved with physical developers, which are solutions that contain a source of silver(I) plus a reducing agent. When the marker is present in extremely small quantities, prolonged incubation in the developer is required and unwanted background staining in the form of type III argyrophilia becomes problematic. Suppression of type III argyrophilia can be achieved by metal-catalyzed oxidation using the copper/H₂O₂ system, which normally is applied immediately prior to amplification. Because there is no reason, in principle, why metal-catalyzed oxidation should not be employed at earlier stages in the immunohistochemical staining procedure, we investigated whether earlier oxidation might confer any advantages over the traditional methodology. Immunocolloidal gold combined with two light insensitive physical developers was chosen as the model system, because visualization by light microscopy requires extended periods in the developers. Moreover, the system does not suffer from problems concerning endogenous enzyme- or non-enzyme-catalyzed marker deposition. Applying metal-catalyzed oxidation at each stage of the immunohistochemical procedure revealed that the technique could be employed successfully prior to staining, but not following the primary or secondary antibodies. In the latter cases, specific immunolocalization was lost entirely and only generalized nonspecific staining was seen. A limited investigation into the mechanism of metal-catalyzed oxidation of aldehyde fixed tissue sections suggested that it involved the formation of aldehyde groups. We suggest that the application of metal-catalyzed oxidation prior to immunohistochemical staining would have the advantages of both suppressing type III argyrophilia and inhibiting unwanted endogenous peroxidase activity. We also suggest that metal-catalyzed oxidation might reduce the affinity of tissue for other transition metals, such as copper, whose potential for improving marker amplification techniques has been demonstrated previously in dot-blot model systems.