Whole-mount Immunolabeling of Cochlear Turns

Whole-mount immunostaining and imaging was performed as previously described (Vyas et al., 2019 (link)). Briefly, grossly dissected inner ears were perfused with 4% paraformaldehyde (PFA; Electron Microscopy Sciences) in 1× PBS (Quality Biological Inc.) through the round window, then postfixed for 30 min on a 3D-Rotator. Mature inner ears were washed (3 × 30 min) in PBS, then decalcified in 125 mm EDTA (Quality Biological Inc.) in PBS for 2 h before dissection into turns of the organ of Corti. Postnatal day 7–10 (P7–10) inner ears were not decalcified before washing in PBS (3 × 30 min) and dissection into turns of the organ of Corti. Cochlear turns were immunolabeled with Goat anti-GFP (Sicgen, catalog #A32814, RRID:AB_2333099) to label GCaMP6f protein. Donkey anti-goat Alexa Fluor 488 (Invitrogen, catalog #AB0020-200, RRID:AB_2762838) was used along with Alexa Fluor 647-conjugated phalloidin (Thermofisher Scientific, catalog #A22287, RRID:AB_2620155) and 4'6-diamidine-2'-phenylindole dihydrochloride (DAPI; Roche Molecular Systems) for GCaMP6f, hair cells, and nuclei, respectively (Fig. 1). Cochlear turns were mounted using ProLong Fade Gold Antifade Mountant (Thermofisher Scientific, catalog #P36930) and imaged on a 700 LSM Zeiss confocal microscope.