Immunostaining of intermediate filament proteins in paraffin sections. Evaluation of optimal protease treatment to improve the immunoreactivity

Different protocols of protease (pepsin) treatment were compared in the immunostaining for intermediate filament (IF) proteins in formaldehyde-fixed and paraffin-embedded tissues. Without protease treatment, the immunoreactivity for all IF proteins was poor in such material. Appropriate pepsin pretreatment improved the immunoreactivity of formaldehyde-fixed tissues for all types of IF proteins, except for the 68 K neurofilament protein, which could not be immunostained with the antibody used. The optimal time for the pepsin treatment was varying in different tissues, and too long treatment caused progressive loss of immunostaining. Thus, for instance when demonstrating cytokeratins, renal adenocarcinomas were more sensitive to protease and needed a shorter treatment than other carcinomas. Therefore, a nonoptimal protease treatment protocol may cause false negative results and false cell type selective IF immunostaining. Prolonged fixation made it necessary to prolong the protease treatment. In tissues fixed up to four years in formalin, cytokeratin immunoreactivity could still be restored by a long pepsin treatment (up to 2-3 hours). For most tissues fixed for 24 hours in formaldehyde, an optimal protocol was the following: 0.05% pepsin (2 U/ml) in HCl, pH 1.8, at +37 degrees C for 20-30 minutes. The protease treatment did not produce false positive results. Alcohol-fixed material was good for IF immunostaining without any protease treatment, but such tissue blocks mostly lost the immunoreactivity during long term storage.