Silver paint

Animals were euthanized and excised inner ears were fixed overnight in 2.5 % gluteraldehyde in 0.1 M phosphate buffer (Sigma-Aldrich). Fixed ears were decalcified for 48 hours in 4.3 % EDTA in 0.1 M phosphate buffer (Sigma-Aldrich). Fine dissection was performed to reveal the organ of Corti, before osmium tetroxide (Agar Scientific)–thiocarbohydrazide (Fluka) processing (adapted from Hunter-Duvar [18 (link)]) was carried out. The inner ears were then dehydrated through increasing strength ethanol solutions (Fisher Scientific) and critical point dried using an Emitech K850 (EM Technologies Ltd). The specimens were then mounted on stubs using silver paint (Agar Scientific) and sputter coated with platinum using a Quorum Q150R S sputter coater (Quorum Technologies). Prepared cochlea were visualised with a JEOL LSM-6010 (Jeol Ltd) scanning electron microscope. Hair cell stereocilia bundle counts were performed by counting the number of adjacent inner hair cells (IHCs) and outer hair cells (OHCs) to ten pillar cells. For the analysis the cochlea was divided into three separate regions (turns), apical (<180° from apex), mid (180–450° from apex), and basal (450–630° from apex). Four ears (one ear per mouse) were analysed for each genotype at each region.

The area of interest with the selected cells was trimmed from the plastic block and mounted onto a pin using conductive epoxy glue (model 2400; CircuitWorks, Kennesaw, GA, USA). The trimmed block was further trimmed as a pyramid and its sides were covered with silver paint (Agar Scientific Ltd., Stansted, UK). To improve conductivity, the whole assembly was platinum-coated using Quorum Q150TS (Quorum Technologies, Laughton, UK). SB-EM data sets were acquired with a FEG-SEM Quanta 250 (Thermo Fisher Scientific, FEI, Hillsboro, OR, USA), using a backscattered electron detector (Gatan Inc., Pleasanton, CA, USA) with 2.5-kV beam voltage, a spot size of 2.9, and a pressure of 0.15 Torr. The block faces were cut with 50-nm increments and imaged with XY resolution of 25 nm per pixel. The collected 16-bit images were processed for segmentation using an open-source software Microscopy Image Browser [51 (link)] as follows: (a) individual images were combined into 3D stacks; (b) the combined 3D-stack was aligned; (c) the contrast for the whole stack was adjusted, and (d) the images were converted to the 8-bit format.

Each group had two randomly selected samples processed for SEM (Quanta FEG 400; FEI Co., US) analysis. The root halves were attached to blades made of aluminum (Silverpaint; Agar Scientific Ltd., Stansted, Essex, UK) and then they were dried out using ethanol solutions. The samples were coated and examined under × 2000 magnification.

Inner ears were collected and processed for SEM, as previously described (47 (link)). Briefly, mice were culled by cervical dislocation and inner ears were removed and fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer. Following decalcification in 4.3% EDTA, cochleae were sub-dissected to expose the sensory epithelia and subjected to processing with an alternate incubation in 1% osmium tetroxide and 1% thiocarbohydrazide, before dehydration in increasing concentrations of ethanol. Samples were critical point dried using an Emitech K850 (EM Technologies Ltd), mounted on stubs using silver paint (Agar Scientific) and sputter coated with platinum using a Quorum Q150R S (Quorum Technologies). Cochleae were examined using a JEOL JSM-6010LV scanning electron microscope. Hair cell stereocilia bundle counts were performed by counting the number of outer hair cell and inner hair cell bundles adjacent to ten pillar cells in the apical (<180° from apex), mid (180–450° from apex) and basal (> 450° from apex) regions of the cochlea. At least six ears (one ear per mouse) were analysed for each genotype.