Autosamdri 815a

Myo3a mutant mice and wild-type mice were anesthetized using pentobarbital sodium and perfused with 4% PFA. The cochleae from Myo3a mutant and wild-type mice were removed, fixed with 2.5% glutaraldehyde in 0.1 M PBS at 4°C overnight, and decalcified in 10% EDTA [31 (link)]. The cochleae were dissected out from the temporal bone, and the stria vascularis, Reissner's membrane, and tectorial membrane were removed [32 (link)]. The organ of Corti was exposed and postfixed in 1% osmium tetroxide in 0.1 M phosphate buffer [33 (link)] for 2 h before dehydration in an ethanol series and critical drying point in an Autosamdri-815A (Tousimis) [32 (link)]. Samples were mounted with carbon tape, coated with gold, and imaged with a JEOL 7000 field emission gun scanning electron microscope.

We referenced Casado et al.’s method [49 (link)] for sample preparation and made improvements. At the equator of the grapefruit, we used a blade to trim the waxy peel to about 2 mm × 2 mm × 1 mm, and fixed the peel in 2.5% glutaraldehyde. The samples were dried with a critical point dryer (Autosamdri-815A, Tousimis, Rockville, MD, USA) and attached by a conductive tape to a sampling table. A gold particle was coated with Hitachi MC1000 and examined with a Hitachi SU8020 field emission scanning electron microscope.

Strips of the rabbit skin were cut using surgical blade and a steel ruler (the latter to keep the cuts straight). The strips were first immersed in 2.5% glutaraldehyde for 3 h to fix the structure, and dehydrated with an ascending ethanol series (30, 50, 70, 90, 95 and 100 vol.% twice) while preventing shrinkage due to dehydration. The strips were fractured using forceps immediately after being immersed in liquid nitrogen. The fractured samples were immersed in ethanol and dried in a critical point dryer (Auto Samdri 815A, Tousimis). The dried fracture surfaces were then sputter coated with iridium using an Emitech K575X sputter coater (Quorum Technologies Ltd.) and examined by FEI SFEG ultra-high resolution SEM (FEI, Hillsboro).
Samples were also observed under wet conditions using an in situ SEM (Hitachi S-4300SE/N SEM (Hitachi America) during the tension testing. However, owing to the wet condition of the skin sample, high resolution could not be obtained. Some stretched samples in different tensile stages (toe, heel, linear and fracture) were prepared using the similar SEM sample preparation procedure (structure fixing, dehydration and critical point drying) and observed using FEI SFEG ultra-high-resolution SEM. All structure-fixed samples, which were tensile tested, were subsequently characterized in the SEM.

The experiments were performed as described (Zhao et al., 2016 (link); Liu et al., 2018 (link)). In brief, inner ears from P7 pups were dissected in a fixative containing 2.5% glutaraldehyde and 4% formaldehyde. Samples were then fixed for 1 h at RT and dissected to remove the stria vascularis, Reissner’s membrane, and tectorial membrane. After being post-fixed in the same fixative overnight at 4°C, samples were fixed with 1% OsO₄ for 1 h at room temperature. Then samples were serially dehydrated in ethanol, dried in a critical point drier (Autosamdri-815A, Tousimis, Rockville, MD, USA), finely dissected, and mounted on aluminum stubs. Samples were then coated by gold. Images were captured using a JEOL 7800F scanning electron microscope (Jeol, Tokyo, Japan). At least three animals representative of each experimental paradigm were analyzed.
To measure the width of the tallest row of stereocilia in inner hair cells, single hair cells were imaged at high magnification (×~20,000) using a JEOL 7800F scanning electron microscope. Then, the width of the tallest row of stereocilia at about 1/3 of the height from the top was measured using ImageJ (NIH, Bethesda, MD, USA).