Cochleae were fixed with 4% paraformaldehyde in phosphate‐buffered saline (1 h). Sections of the cochlea were cryosectioned following 0.12 M EDTA decalcification. After incubation of sections for 1 h in goat serum dilution buffer (16% normal goat serum, 450 mM NaCl, 0.6% Triton X‐100, 20 mM phosphate buffer, pH 7.4), primary antibodies were applied over night at 4°C. The following antibodies were used as follows: chicken anti‐GFP (catalog no.: ab13970, Abcam, 1:500) and guinea pig anti‐parvalbumin (catalog no.: 195004, Synaptic Systems, 1:300). Thereafter, secondary AlexaFluor‐labeled antibodies (goat‐anti‐chicken 488 IgG (H + L), catalog no.: A‐11039, Thermo Fisher Scientific, 1:200; goat‐anti guinea pig 568 IgG (H + L), catalog no. A1107, Thermo Fisher Scientific, 1:200) were applied for 1 h at room temperature. Confocal images were collected using a SP5 microscope (Leica) and processed in ImageJ. Expression was considered positive when anti‐GFP immunofluorescence in a given cell (marked by anti‐parvalbumin immunofluorescence) was found to be higher than 3xSD above the background fluorescence.

For analysis of ChR distribution, line profiles (length: 7.5 µm, width: 3 pixels) were aligned to the NG (visually aligned on the cytosol) and SGN (approximated as the position for which parvalbumin immunofluorescence rose to 50% of its intracellular value) cell membrane. The line profiles were oriented perpendicular to the cell edge. For membrane/intracellular expression ratio, a maximum peak detection was performed for membranous area (defined as 0 µm) and for intracellular area (defined as 1.12 µm).

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