IMMUNOSTAINING OF CHAIN-SPECIFIC KERATINS ON FORMALIN-FIXED, PARAFFIN-EMBEDDED TISSUES - A COMPARISON OF VARIOUS ANTIGEN RETRIEVAL-SYSTEMS USING MICROWAVE-HEATING AND PROTEOLYTIC PRETREATMENTS

The use of chain-specific monoclonal antibodies (MAbs) against keratin s in pathology is hampered by their limited staining on formalin-fixed , paraffin-embedded tissue. In the present study, various treatments b efore immunohistochemistry on paraffin sections were compared, includi ng proteolytic enzymes and microwave antigen retrieval in various solu tions. Sections of normal cervical and skin tissue were stained in a t hree-step immunoperoxidase method, employing a broad panel of MAbs aga inst chain-specific keratins 4, 5, 7, 8, 10, 13, 14, 17, 18, 19 and pa nkeratin. Using microwave heating, Target Unmasking Fluid (TUF), Antig en Retrieval Solution (ARS), a simple detergent solution (DET), PBS, a nd distilled water (MiQ) were compared. Microwave heating in PBS or Mi Q strongly improved staining results. Moreover, microwave pre-treatmen t 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 morphol ogy remained optimal, and only in a very limited number of MAbs did im munoreactivity on paraffin sections fail to be restored. Proteolytic p re-treatment with trypsin, pepsin, or pronase gave moderate to strong staining with some of the MAbs. Other MAbs, for which microwave pretre atment was able to restore the loss of immunoreactivity, failed to giv e appropriate staining with proteolytic pretreatment. Our results show that microwave heating in either TUF or a simple detergent solution b efore immunohistochemistry is a reliable method for antigen retrieval tissues.