Ab150135

Cells were fixed in methyl alcohol and permeabilized with 0.3% Triton X-100 in phosphate-buffered saline (PBS). After blocking in 10% bovine serum albumin (BSA), cells were incubated overnight at 4 °C with the following primary antibodies: anti-GFAP (MAB360; EMD Millipore, Burlington, MA, USA), anti-aldh1L1 (NBP2-50045; Novus Biologicals, Littleton, CO, USA), anti-nestin (ab92391; Abcam), anti-Oct4 (ab18976; Abcam), anti-neuronal nuclear protein (NeuN) (MAB377; EMD Millipore), anti-MAP2 (M9942; Sigma-Aldrich, St. Louis, MO, USA and ab32454; Abcam), anti-ionized calcium-binding adaptor molecule 1 (Iba1) (PA5-27436; Invitrogen, Carlsbad, CA, USA and NB100-1028; Novus Biologicals), anti-bromodeoxyuridine (BrdU) (B5002; Abcam), anti-Ki67 (ab15580; Abcam), anti-BDNF (ab108319; Abcam), and anti-nerve growth factor (NGF) (ab52918; Abcam). After treatment with primary antibodies, the cells were washed with PBS and then incubated with secondary antibodies conjugated with Alexa488, Alexa555 (A11001, A22428; Invitrogen), and Alexa647 (ab150135; Abcam) for 1.5 h at room temperature. After washing with PBS, immunostained cells were mounted with VECTASHIELD^® Antifade mounting medium with DAPI (H-1200; Vector Laboratories, Burlingame, CA, USA) and observed with a confocal microscope (FV31-S; Olympus, Tokyo, Japan).

The cochlea harvested from the in vitro experiment was sliced according to the method mentioned in HE staining. 0.1% Triton X-100 (×100; Sigma–Aldrich) was dropped on the fixed sections. Following 15 min of permeabilization, 5% normal goat serum (SL038; Solarbio, China) was used to seal the tissue sections. After 1 h, the anti-COX26 antibody (ab59020; Abcam, UK) diluted 200 times and covered with the cochlea sections overnight (4°C). After the surface liquid was sucked, the cochlea sections were further reacted with the labeled Donkey Anti-Goat antibody (fluorescently labeled Alexa Fluor^® 647; dilution ratio: 1/500; ab150135; Abcam) for 1 h, followed by being stained with DAPI staining and mounted with fluorescent mounting fluid. The stained sections were observed under a microscope at a magnification of ×400, and the expression of COX26 in the Corti’s organ was detected.

Osteoblasts were seeded onto a 35 mm Glass Bottom Dish and allowed to equilibrate for 12 h. Osteoblasts were then cultured for 24 h with fresh 10% FBS α-MEM containing either 10 ng/ml FGF7 or PBS solution. After the treatments, the cells were washed three times for 2 min and fixed with 4% paraformaldehyde for 10 min, followed by three rinsed with PBS. Then they were permeabilized with 0.25% Triton X-100 for 5 min, washed with PBS. Afterward, the cells were blocked with 5% bovine serum albumin for 60 min, washed with PBS, followed by the addition of E11 antibody (1:200) (R&D Systems, AF3244) and connexin43 antibody (1:200) (Abcam, ab11370) and then incubated overnight at 4 °C. Following incubation, the cells were washed three times for 15 min with PBS. Either Donkey anti-goat IgG (Abcam, ab150135) or Donkey anti-rabbit IgG (Abcam, ab150075) was added and incubated for 2h at room temperature. Then, FITC-conjugated phalloidin (Invitrogen, CA, USA) in PBS was added for double staining and incubated overnight at 4 °C. Followed incubation, the cells were washed three times for 5 min and then stained by DAPI (Sigma, Aldrich, Louis, MO, USA) for nucleus staining. The cell images were captured using a modified confocal laser scanning microscope. (A1R MP+, Nikon, Tokyo, Japan and Olympus, FV3000, Japan) and analyzed with Image-Pro Plus Software 6.0.