Anti prestin

The 4 inner ears of 2 very fresh individuals (sampled and fixed within 18 hours postmortem) were collected following earlier published methods.⁴³ The ears were transported to the University of British Columbia (UBC), Canada, for analysis, with appropriate CITES permits (14NL220354/12, 14CA01694/CWHQ-1, 16NL231424/12, and 16CA02279/CHWQ). The inner ears were processed for scanning electron microscopy (SEM) and histopathology in the first individual, and for SEM and immunofluorescence for the second individual, following previously optimized protocols.^{44 (link)–47 (link, link, link)} The sensory cells of the organ of Corti were labelled with anti-prestin (Santa Cruz, SC-22692, 1:200) and anti-myosin VI (Proteus Biosciences, 25-6791, 1:500) antibodies and type I afferent innervation was labelled with anti-neurofilament 200KD (Sigma-Aldrich N0142, 1:400). Nuclei were counterstained with DAPI (4′,6-diamidino-2′-phenylindole, dihydrochloride; Thermo Scientific, 62247). The inner ears were observed using an S-4700 SEM and an Olympus FV1000 confocal microscope at the UBC Bioimaging Facility.

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.