Quantitation of stereocilia dimensions in transduction and Myo15a mutants

Transduction mutant length and width data.xlsx contains data for <i>Tmie</i> KO and <i>Tmc</i> DKO mice at P7.5 and P21.5.<br><br>sh2 length and width data.xlsx contains data for <i>Myo15a^sh2</i> mice at P8.5.<br><br><br>For analyzing length and width of apical IHC stereocilia of <i>Tmie</i>, <i>Tmc1;Tmc2 </i>and <i>Myo15a^sh2</i> mutant mice at P7.5 and P21.5, z-stack images of phalloidin-stained IHC stereocilia from cochleas of mutant mice were obtained using identical image acquisition parameters. Images were acquired on a ZEISS Elyra PS.1/LSM 710 Airyscan combination setup, using a 63x 1.4 NA Plan-Apochromat or a 100x 1.46 NA alpha Plan-Apochromat lens, integrated under ZEN 2.1 software. Raw data processing for Airyscan-acquired images was performed using manufacturer-implemented automated settings. At least 2-4 cochleas were used for each genotype. Images containing hair bundles that were upright were selected for generating x-z reslices of stereocilia with the ‘Reslice’ function in Fiji. Reslices were chosen with views of stereocilia rows 1 and 2; we chose central stereocilia in most bundles. Stereocilia lengths were measured individually in Fiji by manually drawing a vertical line from the top of a stereocilium to the point of insertion in the cuticular plate. For measuring stereocilia widths, horizontal lines were manually drawn at 50% of the bundle height for both rows 1 and 2.<br>