Optimizing immunofluorescence for the testing of autoantibodies in inner ear disorders

A method is described for the immunofluorescent testing of autoantibodies in the sera of patients with vestibulo-cochlear disorders, using fixed decalcified inner ear tissue preparations from guinea pigs. Fixation in cold ethanol and decalcification in EDTa at 4 degrees C preserved the immunological reactivity of the inner ear tissue in use and allowed excellent delineation of its structural details. Counterstaining of the sections with Evans-blue dye aided in a better identification of the inner ear anatomical structures and in a more precise location of the antibodies present. By mounting the sections in p-phenylenediamine-PBS-glycerin solution, the rapid extinction of the fluorescence seen during photomicroscopy was retarded and the intensity of the immunofluorescence was enhanced. Preservation of the slides for documentation was improved by fixing the intermediate layer of the antigen and the labeled antibody in cold ethanol. The inner ear tissue preparations retained their fluorescent staining up to 6 months. Our method is convenient for screening patients with inner ear disorders for autoantibodies against the different cellular elements and for testing the possible presence of antibodies against the inner ear tissue. So far, we believe that the antibodies detected are not tissue (inner ear) specific.