Quantitation of stereocilia dimensions during C57BL/6 development

For analyzing length and width of apical IHC stereocilia during development, z-stack images of phalloidin-stained IHC stereocilia from cochleas of C57BL/6J 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 developmental ages E18.5 (embryonic), P0.5, P4.5, P7.5, P10.5, P12.5, P15.5, P19.5 (post-natal). For each developmental age, images containing hair bundles that were upright were selected for generating x-z reslices (Fig. S1C) 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.