Immunofluorescence Staining of Disc Cells

Our previous study also described this method in detailly [2 (link)]. After intervertebral disc sections or NP cells were well prepared, 0.1% Triton X-100 was employed to permeate samples for 5 min. Then, samples were blocked with 3-5% BSA for 60 min at 37°C. Then, the samples were incubated using primary antibody against γH2AX (GB111841, 1 : 200, Servicebio, Wuhan, China), aggrecan (GB11373, 1 : 500, Servicebio, Wuhan, China), collagen type II (GB14073, 1 : 500, Servicebio, Wuhan, China), iNOS (GB11119, 1 : 1000, Servicebio, Wuhan, China), collagen type I (GB11022, 1 : 500, Servicebio, Wuhan, China), NLRP3 (GB11300, 1 : 1000, Servicebio, Wuhan, China), CD206 (#24595, 1 : 800, Cell Signaling Technology, Inc. USA), MMP3 (GB11131, 1 : 500, Servicebio, Wuhan, China), AGT (ab108334, 1 : 200, Abcam, USA), Nrf2 (340675, 1 : 500, Zenbio, Chengdu, China), and p65 (GB11142, 1 : 200, Servicebio, Wuhan, China) at 4°C overnight. At the second day, samples were treated with fluorescence secondary antibody (GB22303, GB21301, Servicebio, Wuhan, China) for 1 h in the dark room. The nuclei were staining with DAPI solution (G1012-100ML, Servicebio, Wuhan, China). The fluorescence was detected using fluorescence microscope (Olympus, Japan).