Technical report: immunofluorescence and TUNEL staining of celloidin embedded human temporal bone tissues

The large archival human temporal bone collections of the world have been fixed in formalin and embedded in celloidin. These treatments have created challenges to the use of contemporary probes, which are routinely used in the evaluation of fresh and frozen tissues, for the analysis of archival temporal bone tissues. Formalin alters the configuration of proteins and can obscure antigens by modifying the epitopes recognized by antibodies. Celloidin embedding provides superior support of the delicate membranous structures of the inner ear to maintain tissue integrity during sectioning, however, inadequate removal of celloidin may limit tissue permeability resulting in poor penetration of large molecules. Methods are described in this manuscript that have allowed reproducible immunofluorescence and TUNEL (terminal deoxynucleotidyl transferase mediated dUTP nick end labeling) staining results in these archival tissues. To our knowledge, successful immunofluorescence staining of type I collagen, immunofluorescence staining of cytochrome c oxidase subunit III (COX III), and TUNEL staining in archival human temporal bone tissues with confocal microscopy has not been previously reported. These results demonstrate the utility of developing techniques to evaluate the existing collections of archival temporal bones which remain our greatest source of tissue for investigating the causes of ear diseases.