Sigma vp hd field emission sem

Adult mice were killed via CO₂ asphyxiation; animals were perfused intracardially, with 2.5% glutaraldehyde + 2% paraformaldehyde. The cochlea was dissected and a piece of bone was removed from the apex to create an opening. The fixative (2.5% glutaraldehyde, in 0.1 M cacodylate buffer, with 3 mM CaCl2) was perfused through the round window and the apical opening. Cochleae were incubated in fixative overnight at 4 °C, then were decalcified in 4.13% EDTA, pH 7.3, for 10 days at room temperature. After the tectorial membrane was removed, the organ of Corti was dissected from the cochlea and was post-fixed in 1% OsO₄, 0.1 M cacodylate buffer, and 3 mM CaCl₂. The tissue was then processed according to the thiocarbohydrazide-OsO₄ protocol^{63 (link)}. Cochleae were then dehydrated through a series of graded ethanol incubations, critical point dried, and mounted on stubs. After sputter coating with gold, they were imaged on a Zeiss Sigma VP HD field emission SEM using the secondary electron detector.

Adult mice were euthanized by CO₂ asphyxiation before intracardiac perfusion with 2.5% glutaraldehyde (Electron Microscopy Sciences, Hatfield, PA) and 2% PFA. The otic capsule was dissected and incubated in postfixation buffer at 4 °C overnight (2.5% glutaraldehyde, in 0.1 M cacodylate buffer, with 3 mM CaCl₂). For neonatal mouse pups, the samples were dissected and treated with postfixation buffer immediately. The otic capsules from adult mice were incubated for 2 weeks in 4.13% EDTA for decalcification and then further dissected to expose the organ of Corti. Samples underwent the OTOTO procedure^{68 (link)} and dehydrated by using gradient ethanol and critical point drying. After sputter coating with platinum, the samples were imaged on Zeiss Sigma VP HD field emission SEM using the in-lens secondary electron detector.