AN EVALUATION OF ENZYMATIC AND HEAT EPITOPE RETRIEVAL METHODS FOR THEIMMUNOHISTOCHEMICAL STAINING OF THE INTERMEDIATE FILAMENTS

The ability to accurately demonstrate intermediate filament proteins ( IFs) in formalin-fixed tissues is of great importance in the histodiag nosis of neoplasms owing to the highly conserved and cell lineage-spec ific pattern of expression of these proteins. In this study, we evalua ted the use of enzymatic and heat epitope retrieval methods for improv ing the immunoreactivity of the Ifs fixed at different time intervals in formalin. Normal tissue specimens were fixed in formalin for 4, 24, and 48 h and in 70% ethanol as controls. The Ifs were reacted with th e following monoclonal antibodies: anti-cytokeratin antibodies KA4 (re active with cytokeratins 14, 15, 16, and 19) and 10.11 (reactive with cytokeratins 8 and 18), anti-vimentin, antidesmin, anti-glial fibrilla ry acidic protein (GFAP, polyclonal), and anti-neurofilament (NF). All IFs had marked loss of reactivity with each monoclonal IF antibody af ter 24-48 h of fixation, except GFAP, which retained its reactivity wi th the polyclonal GFAP antibody used. The cytokeratin immunoreactivity for both anti-cytokeratin antibodies, KA4 and 10.11, was completely r ecovered by predigestion with protease I but was less effectively enha nced by microwave heating. The other Ifs tested greatly benefited from heat epitope retrieval in citrate buffer (0.01 mol/L) at pH 6. Protea se I worked less well for these Ifs and completely destroyed vimentin and desmin reactivity. Unexpectedly, microwave heating of fixed specim ens of kidney and liver in Tris buffer (0.5 mol/L) at pH 10 produced a strong, granular staining artifact that was due to the enhancement of avidin binding to endogenous biotin. This artifactual staining was su ccessfully blocked by application of purified avidin before the additi on of avidin-biotin-peroxidase complex. In conclusion this study reemp hasizes the importance of testing individual antibodies using the vari ous conditions of fixation and epitope recovery.