Preparation of cochlear explants and drug exposure

Cochleae were removed from P3 FVB mice and placed in precooled sterile HBSS (MilliporeSigma, H6648). The organ of Corti was dissected and maintained in culture medium (DMEM/F12 containing 1% FBS, 2% B-27 supplement, 1% N-2 supplement, and 50 μg/mL ampicillin). After overnight incubation at 37°C in 5% CO₂, the medium was replaced with growth media with or without Qx28 (10 nM–1 mM). After 1-hour incubation, fresh media containing 100 μM gentamicin (MilliporeSigma, G1397) or 8 μM CDDP (Accord, NDC16729-288-11) was added (with or without Qx28). Explants were incubated for 24 hours with the gentamicin or for 48 hours with CDDP, PFA fixed for 10 minutes, and then processed for immunohistochemistry. The primary antibody incubation was performed overnight at 4°C, followed by secondary antibody (donkey anti-rabbit Alexa Fluor 594, catalog A32754, Thermo Fisher Scientific) incubation for 2 hours at room temperature. Rabbit anti–myosin VI (catalog 25-6791, Proteus Bioscience) was used at a 1:400 dilution. Confocal imaging was performed using a Zeiss LSM 700 confocal system with a 40× oil objective; 200 μm regions from the middle turn were photographed, and the number of intact OHCs in each 30 inner hair cells was counted.