Limitation of microwave treatment for double immunolabelling with antibodies of the same species and isotype

Microwave treatment (MW) involves completely blocking contaminating staining in the double-labelling technique, using primary monoclonal antibodies from the same species and the same isotype as well as the same secondary antibody (ab). However, we noticed some limitations when locating proliferating cell types in cryostat and paraffin sections using the advantages presented by MW. Control experiments have shown that MW does not diminish contaminating staining when cytoplasmic (desmin, ASM-1) or nuclear (Ki-67) antigens have been labelled with antibodies in the first round of immunolabelling. In contrast to the cell surface antigen, CD18, where the primary ab had to be crosslinked by a secondary ab to obtain contaminating staining, this was observed for the detection of cytoplasmic or nuclear antigens only labelled with a primary ab. In conclusion, for double immunolabelling with abs from the same species and the same isotype, MW is not able to completely abolish contaminating staining.