Sp5 confocal fluorescence microscope

After fixation, cochlear samples were blocked with 10% normal donkey serum in 10 mM PBS (pH 7.4) with 0.3% Triton-X100 for 1 h at room temperature (RT) and then incubated with primary antibody overnight at 4°C. The next day the tissues were incubated for 2 h at 4°C with 488- or 594-conjugated donkey secondary antibody (1:500 dilution, Invitrogen) and 4,6-diamidino-2-phenylindole (DAPI, 1:800 dilution, Sigma-Aldrich). Omission of primary antibody served as the negative control. The following primary antibodies were used: anti-myosin VIIA (myo7a) (1:500 dilution, cat. #25-6790 Proteus BioSciences), and anti-cleaved caspase-3 (1:1000 dilution, cat. #9579S, Cell Signaling Technology).
Cochleae were dissected into apical, middle, and basal turns and images were taken using a Leica SP5 confocal fluorescence microscope (Leica, Germany).

Immunofluorescence was performed as reported previously (Jiang et al., 2014 (link)). Fixed tissues were blocked with 10% donkey serum in 10 mM PBS (pH 7.4) with 1% Triton X-100 (Sangon Biotech) for 1 h at room temperature and then incubated with primary antibodies overnight at 4°C in a humidified chamber. The following day, tissues were rinsed with PBS and then incubated with secondary antibodies for 1 h at room temperature. After washing with PBS, tissues were mounted in antifade fluorescence mounting medium (DAKO) and coverslipped. The primary antibodies included anti-Lgr6 (1:500 dilution, Santa Cruz Biotechnology), anti-myosin 7a (1:1000 dilution, Proteus Bioscience), anti-GFP (1:1000 dilution, Abcam), and anti-Sox2 (1:500 dilution, Santa Cruz Biotechnology). The secondary antibodies were conjugated with FITC, Cy3, or Cy5 (1:500 dilution, Jackson ImmunoResearch). The nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI, 1:800 dilution, Sigma-Aldrich) for 15 min at room temperature. Negative control experiments were performed as above by omitting the primary antibodies. Cochleae were dissected into the apical, middle, and basal turns, and photographs were taken using a Leica SP5 confocal fluorescence microscope (Leica) and analyzed with Photoshop CS4 (Adobe Systems).

For IF assays, cells were cultured on glass coverslips. After different treatments, cells were washed with PBS and fixed with 4% PFA. The cells were permeabilized with 0.2% Triton X-100 followed by blocking in 1% BSA. Primary antibodies were incubated overnight at 4 °C. The cells were washed again for three times in PBS and then incubated with fluorochrome-conjugated second antibody for 1 h at 37 °C. For further nuclear staining, cells were incubated with 4′,6′-diamidino-2-phenylindole (DAPI). Finally, the fluorescence-labeled cells were mounted with ProLong antifade reagent (Invitrogen) and photographed using a Leica SP5 confocal fluorescence microscope (Leica, Wetzlar, Germany).