Ab150107

Fluorescence-activated cell sorting (FACS) was performed as previously described^{8 (link)–10 (link),27 (link),28 (link)}. Briefly, cells adherent to culture dishes were first washed with DPBS and trypsinized with 0.25% trypsin/EDTA. Cells were then fixed with fixation buffer (BD Biosciences), washed with Perm/Wash buffer (BD Biosciences) and then incubated with mouse monoclonal anti-cardiac troponin T (cTnT) antibody (ab8295; Abcam) in Perm/Wash buffer. These cells were then incubated with donkey anti-mouse Alexa Fluor 647 (ab150107; Invitrogen™), washed 3× with Perm/Wash buffer again, and further analyzed for cTnT expression using a LSR Fortessa cell sorter (BD Biosciences) with FlowJo software (FlowJo, LLC, Ashland, Ore) and Diva software (version 6.0).

The cochleae were placed in the medium containing 3 mM FM1-43 (Thermo Fisher, F35355) for 90 s and washed three times in PBS (pH 7.2). The cochleae then were dissected and fixed with 4% polyoxymethylene for 1 h and permeabilized with 0.5% Triton X-100 for 1 hour. The sensory epithelia were then incubated with the following primary antibodies overnight at 4 °C: anti-LIMK1 (Santa Cruz, sc-8387); anti-LIMK2 (Santa Cruz, sc-5577); anti-MYO7A (Proteus Bioscience, 25-6790); anti-SOX2 (Santa Cruz, sc-17320, sc-365823); anti-CtBP2 (C-terminal-binding protein 2), anti-IgG1 (BD Biosciences, 612044), anti-PSD95 IgG2a (Millipore, MAB1596), anti-cofilin (Abcam, ab42824), anti p-cofilin (Santa Cruz, sc-12912), and anti-prestin (Santa Cruz, sc-22692). Phalloidin (Invitrogen, A34055) was used to stain the actin cytoskeleton, and DAPI was used to label the nuclei. The tissues were washed three times with PBST (0.1 M phosphate buffer, pH 7.2, with 0.1% Triton X-100) and incubated for 1 h (37 °C) with DAPI (Sigma-Aldrich, D9542) and suitable secondary antibody (Abcam: ab150075, ab150105, ab150074, ab150073, ab150107; Invitrogen: A21131, A21124). Finally, the sensory epithelia were mounted on glass slides with Fluoromount-G mounting medium. Images were taken using a Zeiss LSM700 confocal microscope.