Whole Mount Immunostaining of Cochlear Hair Cells

Cochlear whole mounts were dissected and mounted as described previously (76 (link)). Immunofluorescent staining was carried out as reported (77 (link)). The following antibodies/stains at the specified concentrations were used: rabbit anti-Myosin 7A (Myo7A, #25-6790 Proteus Biosciences, Ramona, CA, 1:400), mouse anti-Myosin 7A (Myo7A, #138-1-S, Developmental Studies Hybridoma Bank, Iowa City, IA, 1:10), rabbit anti-GRO alpha (CXCL1) biotin (#NBP1-51188B, Novus Biologicals, Littleton, CO, 1:150), mouse anti-E-Cadherin (#610181 BD Biosciences, San Jose, CA, 1:200), sheep-anti DARC (#PA5-47861 Thermo Fisher Scientific, Waltham, MA, 1:25) Phalloidin (#A22283 Thermo Fisher Scientific, Waltham, MA, 1:200), Alexa Fluor 555 anti-mouse and Alexa Fluor 647 anti-rabbit (#A21422 and #A21245 Thermo Fisher Scientific, Waltham, MA, 1:1,000), Alexa Fluor 488 anti-sheep (#A-11015 Thermo Fisher Scientific, Waltham, MA, 1:400), Streptavidin, Alexa Fluor 488 (#S11223, Thermo Fisher Scientific, Waltham, MA, 1:200). For the quantification of inner and outer hair cell numbers before and after noise exposure, the same confocal microscopy settings were used (Leica SP5, Wetzlar, Germany). Images were obtained with 20x, 40x, and 63x objectives. The z-step size was 1.5–2 μm, and cells were counted over a distance of 300 μm.