Donkey anti rabbit 488

The cochleae from adult mice were dissected from the temporal bones and fixed in 4% paraformaldehyde at 37 °C for 60 min. After three rinses with PBS, the cochleae were soaked in 10% EDTA for 3-day decalcification. The entire cochlear explant was divided into three equal parts presented as the basal, middle, and apical turn. The primary antibody used in our study was an anti-myosin VIIA antibody (1:500 dilution; Proteus Biosciences). After washing three times with PBS to remove the unbound primary antibodies, the samples were incubated for 1 h at 37 °C with the secondary antibody donkey anti-rabbit 488 (1:500 dilution, Invitrogen). Nuclei were counterstained with DAPI (1:800 dilution, Sigma–Aldrich) at 37 °C for 10 min, and the samples were mounted and imaged under a Leica SP8 laser scanning confocal fluorescence microscope (Leica Microsystems, Biberach, Germany).

All sections went through antigen retrieval in boiled sodium citrate buffer and incubated in water bath at 80°C for 20 minutes. The cooled sections were washed with washing buffer (0.3% Triton X-100 in 0.05M PBS buffer) and incubated with blocking buffer (1% bovine serum albumin in washing buffer) for 1 hour. The primary antibody rabbit anti-IBA1 (FUJIFILM) with a concentration of 1:1,000 in blocking buffer was applied to skin sections overnight at 4°C, and secondary antibody donkey antirabbit-488 (1:800, Invitrogen) for 90 minutes at room temperature. The sections were rinsed with washing buffer and incubated with DAPI (1:5,000, Sigma) buffer, washed, and finally mounted on slide with 0.5% gelatine and fluorescent mounting medium and coverslipped.
Z-stack confocal images consisting of 15-image stacks with a 21-µm scanning thickness were acquired by a laser scanning spectral confocal microscope (Zeiss LSM 800, Germany) with a 40X oil lens. To reconstruct an image of the entire region of interest (ROI) in 1 skin section, the tile function was used to stitch all single-field images from the same skin section. A minimum of 2 individual skin sections from the same participant were imaged and used for IBA1 quantification.

Slides were post-fixed at room temperature in 4% PFA for thirty minutes then washed once for five minutes in 1x PBS. Sections were permeabilized with 0.2% Triton-X100 for ten minutes at room temperature, then rinsed in 1x PBS before blocking for thirty minutes at room temperature followed by the primary antibody overnight at 4°C. Primary antibodies: Polyclonal Sheep anti-Mouse PEAR1 (R&D Systems, Cat # AF7607; used at 1:50 in blocking solution); Polyclonal Rabbit anti-Mouse IBA1 (Fujifilm Wako Chemicals, Cat # 019–19741; used at 1:500 in blocking solution). Slides were washed twice for three minutes in wash buffer, then received secondary antibodies for one hour at room temperature in the dark. Secondary antibodies: Donkey anti-Sheep 568 (Abcam, Cat # ab175712; used at 1:1000 in blocking solution); Donkey anti-Rabbit 488 (Invitrogen, Cat # A-21206; used at 1:1000 in blocking solution); Goat anti-Rabbit 647 (Invitrogen, Cat # 21245; used at 1:1000 in blocking solution). Slides were washed twice, three minutes per wash, in wash buffer. Mounting media was applied, a glass coverslips placed on top, and slides were left to dry in the dark at room temperature until imaging.