Mouse anti myosin7a

Inner ears from wild-type and Tecta/Tectb^-/- mice (n = 4 for each experiment) were removed by dissection and fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS, pH 7.4) for 20 min at room temperature. Cochleae were microdissected, rinsed 3 times for 10 min in PBS, and incubated for 1 h at room temperature in PBS supplemented with 5% horse serum (HS) and 0.5% Triton X-100. The samples were then incubated overnight at 37°C with the primary antibody in PBS supplemented with 1% HS. Primary antibodies were: mouse anti-myosin 7a (1:1000, Developmental Studies Hybridoma Bank, #138-1C), rabbit anti-myosin 7a (1:200, Proteus Biosciences, #25-6790), rabbit anti-prestin (1:5000, kindly provided by Robert Fettiplace), rabbit anti-SK2 (1:500, Sigma-Aldrich, P0483) and goat anti-choline acetyltransferase (ChAT, 1:500, Millipore, AB144P). All primary antibodies were labelled with species-appropriate Alexa Fluor secondary antibody for 1 h at 37°C. Samples were then mounted in VECTASHIELD. The z-stack images were captured with a Nikon A1 confocal microscope equipped with Nikon CFI Plan Apo 60X Oil objective in the Light Microscope Facility at the University of Sheffield. Image stacks were processed with Fiji Image Analysis software (ImageJ, NIH and LOCI Laboratory of Optical and Computational Instrumentation, USA).