Mounting solution

Whole-mount immunofluorescence of cochlea was performed as described previously.³⁰ (link) In brief, isolated cochleas were obtained by microdissection. Tissues were fixed by submersion in 4% formaldehyde at 4°C overnight. After washing twice with PBS, the fixed temporal bones were decalcified for 24 h in 10% ethylenediaminetetraacetic acid (EDTA)/PBS. For paraffin sectioning and H&E staining, serial dehydration of the tissues was performed with ethanol and xylene. Then, the tissues were either embedded in paraffin for standard histological examination or dissected into smaller pieces and permeabilized with 0.1% Triton X-100 for whole-mount immunofluorescence. The paraffin blocks were sliced into 5-μm-thick sections in the midmodiolar plane using a microtome (Leica Biosystems, Nußloch, Germany). Deparaffinization was performed on the sections and whole-mounted tissues with a series of washes with xylene, ethanol, and PBS. After incubation in tris-sodium citrate at 95°C for antigen retrieval, the tissues were blocked with 10% donkey serum and incubated with target-specific primary and secondary antibodies at 4°C overnight. The samples were then mounted with mounting solution (Sigma-Aldrich) and viewed under an LSM780 confocal microscope (Zeiss, Jena, Germany).

After sacrificing the mice in a CO₂ chamber, the bilateral temporal bones were dissected and fixed in 4% paraformaldehyde for 24 hours at 4°C after local perfusion with a fixative through the oval and round windows. After fixation, the samples were incubated in 1:3 EDTA (ethylenediaminetetraacetic acid) solution for 24 hours at 4°C for decalcification.
For obtaining a whole mount of the organ of Corti sample, decalcified cochleae were cut in half through the apex–oval window axis. Under optical microscopy, the cochleae’s lateral wall and bony capsule were carefully removed using forceps and microscissors. Other structures were trimmed while preserving the organ of Corti. All cochlear tissues were separated into apical, middle, and basal turns.
The tissues were blocked with 10% donkey serum and incubated with dye-conjugated Ly6C antibody (HK1.4, 128016; Biolegend, San Diego, CA, USA) and/or dye-conjugated F4/80 antibody (BM8, 123110, Biolegend) at 4°C overnight. The samples were then mounted with a mounting solution (Sigma-Aldrich, St. Louis, MO, USA) and viewed under an LSM780 confocal microscope (Zeiss, Jena, Germany). All immunostaining experiments were repeated in four biological replicates per time point.

Tissues from the inner ears of the mice were fixed in 4% paraformaldehyde at 4°C overnight and then washed twice with PBS. Subsequently, the specimens were decalcified in 25% EDTA/PBS for 24 h. Tissues were dehydrated and embedded in paraffin for histology and dissected into segments with the organ of Corti and permeabilized with 0.1% Triton X-100 for whole-mount immunostaining. The paraffin blocks were cut into 5-μm sections using a microtome (Leica Biosystems) and subjected to hematoxylin and eosin (H&E) staining and immunostaining. H&E-stained tissues were examined under a light microscope. For immunostaining, paraffin sections were deparaffinized with xylene, ethanol, and PBS. Thereafter, for antigen retrieval, the sections were incubated in sodium citrate at 95°C for 5 min and then blocked in 10% normal donkey serum for 1 h at room temperature. The tissues were incubated overnight with target-specific primary antibodies at 4°C, washed, and incubated for 1 h at room temperature with the appropriate secondary antibody (Table S1). Finally, the samples were washed and then mounted with mounting solution (Sigma-Aldrich, St. Louis, MO, USA). All images of immunostaining were obtained under a confocal microscope LSM780 (Zeiss Laboratories, Jena, Germany).