Vélez laboratory

All animal procedures were approved by the University of Kentucky Animal Care and Use Committee (protocol 2020-3535). Organ of Corti explants were isolated from C57BL/6 mice at postnatal day 4 in cold Leibovitz’s L-15 medium (cat. # 21083027, Gibco) and held in place by two glass fibers glued to the bottom of a Petri dish. Explants were cultured in high-glucose DMEM (12430062, Gibco) supplemented with 7% FBS (16140071, Gibco) and 10 mg/L ampicillin (171254, Calbiochem) at 37 °C and 5% CO₂, for 24 h in the presence of 62.5 µM gentamicin (G1397, Sigma-Aldrich) or in control conditions. Next, explants were fixed in 4% PFA (15710, Electron Microscopy Sciences) for 24 h at 4 °C. Samples were then permeabilized with 0.5% Triton-X (22142, Electron Microscopy Sciences) for 1 h and blocked with 10% normal goat serum (10000C, Invitrogen) and 0.25% Triton-X for 1 h, at room temperature. Hair cells were labeled with rabbit polyclonal anti-Myosin-VIIA primary antibody (#25-6790, Proteus Biosciences, concentration 1:100) for 24 h at 4 °C followed by labeling with goat anti-rabbit Alexa fluor 488 (A11034, Invitrogen, 1:1000) as a secondary antibody for 3 h at room temperature. Samples were counterstained with 1 unit of rhodamine phalloidin for 30 min at room temperature (R415, Invitrogen), and mounted in ProLong Diamond antifade medium ({"type":"entrez-protein","attrs":{"text":"P36961","term_id":"547831","term_text":"P36961"}}P36961, Invitrogen). Imaging was performed with a Leica SP8 upright confocal microscope equipped with a Leica HCX PL APO 100 × 1.44 NA objective lens. Confocal z-stacks were taken with a voxel size of 114 nm in X and Y, and 500 nm in Z.