Immunostaining of chain-specific keratins on formalin-fixed, paraffin- embedded tissues: A comparison of various antigen retrieval systems using microwave heating and proteolytic pre-treatments

The use of chain-specific monoclonal antibodies (MAbs) against keratins in pathology is hampered by their limited staining on formalin-fixed, paraffin- embedded tissue. In the present study, various treatments before immunohistochemistry on paraffin sections were compared, including proteolytic enzymes and microwave antigen retrieval in various solutions. Sections of normal cervical and skin tissue were stained in a three-step immunoperoxidase method, employing a broad panel of MAbs against chain- specific keratins 4, 5, 7, 8, 10, 13, 14, 17, 18, 19 and pankeratin. Using microwave heating, Target Unmasking Fluid (TUF), Antigen Retrieval Solution (ARS), a simple detergent solution (DET), PBS, and distilled water (MiQ) were compared. Microwave heating in PBS or MiQ strongly improved staining results. Moreover, microwave pre-treatment in TUF or DET gave excellent and specific staining with the majority of MAbs tested, comparable with or even better than staining obtained on frozen sections. Using microwave antigen retrieval, tissue morphology remained optimal, and only in a very limited number of MAbs did immunoreactivity on paraffin sections fail to be restored. Proteolytic pre-treatment with trypsin, pepsin, or pronase gave moderate to strong staining with some of the MAbs. Other MAbs, for which microwave pretreatment was able to restore the loss of immunoreactivity, failed to give appropriate staining with proteolytic pretreatment. Our results show that microwave heating in either TUF or a simple detergent solution before immunohistochemistry is a reliable method for antigen retrieval of chain- specific keratins in formalin-fixed, paraffin-embedded tissues.