Methods for cellular and subcellular visualization of intermediate filament proteins in the human inner ear

The preservation of antigenicity for monoclonal antibodies (mAbs) directed against the five classes of intermediate filament proteins and their subgroups was analysed in inner ear specimens from both early embryonic (6-8 gestation-week-old) human labyrinths and inner ears from newborn CBA/CBA mice. After initial fixation in 2% paraformaldehyde, the specimens were embedded in either polyvinyl alcohol (PVA) or the low viscosity acrylic resin LR White. Both embedding media allowed sectioning at room temperature with a specimen thickness of 0.5-1 microns, which gives a resolution at the subcellular level in the light microscope. Immunoreactivity occurred in the PVA-embedded material, but not in specimens embedded in LR White. However, considerably fewer mAbs showed immunostaining in the PVA-embedded material than in both cryofixed-cryosectioned or paraformaldehyde-fixed-cryosectioned human inner ears. Immunoelectron microscopy using colloidal gold (particles 10 nm in diameter) was successful in the PVA-embedded (but not the LR White-embedded) material.