Scanning Electron Microscopy of Organ of Corti

Organ of Corti explants were dissected at postnatal day 4 (P4) in L-15 medium and fixed with 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.2), supplemented with 2 mM CaCl₂ for 1–2 h at room temperature. For older (P20–120) animals, temporal bones were fixed in 4% formaldehyde for 1 h at room temperature and decalcified in 5% ethylenediaminetetraacetic acid (pH 7.2–7.4) for 3–4 days at 4 °C. After unpeeling cochlear bone and removing the stria vascularis and TM, the cochlear coils were isolated, divided into apical, middle, and basal turns and postfixed with 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.2), supplemented with 2 mM CaCl₂ for 1–2 h at room temperature. Samples were rinsed in 0.1 M cacodylate buffer (pH 7.2) and then in distilled water, dehydrated in an ascending series of ethanol, and critical-point dried from liquid CO₂ (Tousimis Autosamdri 815).
Samples were then mounted on aluminum stubs with carbon conductive tabs and were sputter-coated (EMS 300 T dual head sputter coater) with either 5-nm platinum (for conventional SEM) or 4.0-nm palladium (for immunogold-SEM) as previously described^{19 (link)} and observed in field emission scanning electron microscope (Hitachi S-4800) or focused-ion beam (FIB) scanning electron microscope (FEI Helios 660) using secondary or backscatter electron detectors.