Magellan 400 xhr field emission scanning electron microscope

Inner ears were isolated in WB (0.05 mm HEPES buffer, pH 7.2, 10 mm CaCl₂, 5 mm MgCl₂, and 0.9% NaCl) and fixed in 4% PFA in 0.05 mm HEPES buffer, pH 7.2, 10 mm CaCl₂, 5 mm MgCl₂, and 0.9% NaCl for 30 min at RT. The inner ears were then dissected to remove the stria vascularis and Reissner's and tectorial membranes. The samples were refixed in 2.5% glutaraldehyde and 4% PFA in 0.05 mm HEPES buffer, pH 7.2, 10 mm CaCl₂, 5 mm MgCl₂, and 0.9% NaCl overnight at 4°C, then washed, dehydrated in ethanol (30%, 75%, 100%, and 100%, 5 min incubations), and processed to the critical drying point using Autosamdri-815A (Tousimis). Cochleae were mounted on studs using silver paint and coated with 5 nm of iridium (sputter coater EMS150TS, Electron Microscopy Sciences). Samples were imaged at 5 kV on an FEI Magellan 400 XHR Field Emission Scanning Electron Microscope at the Stanford Nano Shared Facilities. The microscope is periodically calibrated for measurements using a SIRA-type calibration specimen for ultra-high-resolution modes with ≤2% error between 50 and 350k× magnification at our imaging settings.