Injectoporation of Cochlear Explants

Injectoporation was performed as described (Xiong et al., 2014 (link); Liu et al., 2018 (link)). In brief, the organ of Corti was isolated and cultured in DMEM/F12 medium (11330057; Life Technologies) for 2–4 h. Glass electrodes (2-µm diameter) were used to deliver the plasmid (500 ng/µl in 1× HBSS) to the sensory epithelium. A series of three pulses were applied at 1-s intervals with a magnitude of 60 V and duration of 15 ms (BTX ECM 830 square wave electroporator). Cochlear explants were cultured in a 37°C incubator for 2–3 d. Samples were then fixed in 4% formaldehyde for 20 min at room temperature and processed for immunostaining using 6E2 anti-HA antibody (2367, RRID:AB_10691311; Cell Signaling Technology). Whole-mount preparations were imaged on a Deltavision Deconvolution Microscope and processed with SoftWoRx software (Applied Precision).