Biotin labeled secondary antibody

After 21 d of osteogenic differentiation, cells were fixed with 4% paraformaldehyde for 15 min at room temperature and washed with PBS. Cells were then permeabilized with 0.1% Triton-X (Sigma, USA) for 15 min and blocked with 5% BSA for 1 h (Beyotime, China) at room temperature. Cells were incubated overnight at 4°C with the following primary antibodies: OCN (sc30045, Santa Cruz Biotechnology) and OPN (ab91655, Abcam). Cells were then washed with PBS and incubated with a biotin-labeled secondary antibody (ABclonal, China) for 1 h at room temperature. Cells were then stained with diaminobenzidine (DAB) (Beyotime, China) to detect the protein and then visualized using a microscope (Olympus BX51, Japan).

The paraffin sections of the tissues at 5-μm thickness were baked overnight at 37°C, deparaffinized and incubated using 3% hydrogen peroxide for 15 min. The sections were microwaved to heat the tissue antigens, and then cooled at room temperature for 30 min. The tissue was incubated with mouse FEZ1 (1:50), SCOC (1:50), ULK1 (1:50), and NBR1 (1:100) antibodies, and all of which were obtained from Abclonal company in China. Finally, the tissue was incubated with a biotin-labeled secondary antibody (1:500, Abclonal, China) to observe protein expression.