Ultrastructural Analysis of Prestin-KO Mice

Cochlear samples from WT and prestin-KO mice were prepared for electron microscopy as previously described (He et al., 2010 (link)). Briefly, microdissected cochleae were fixed with 4% paraformaldehyde and 0.5% glutaraldehyde, plunge frozen, and transferred to a Leica Biosystems AFS for freeze substitution. Tissues were submerged in 1.5% uranyl acetate in absolute methanol at -90°C for 2 days and then infiltrated with HM20 Lowicryl resin (Electron Microscopy Sciences) over 2 days at -45°C. Resin was polymerized under UV light with temperatures rising from -45 to 0°C across 3 days. Ultrathin sections were produced using a Leica ultramicrotome, collected onto 300-mesh hexagonal Ni grids (Electron Microscopy Sciences), and examined using a 200 kV JEOL 2100 electron microscope equipped with a Gatan Orius 832 CCD camera. Images were acquired with DigitalMicrograph (Gatan). Fiji software was used to crop, rotate and adjust image brightness and contrast.