Collagen 2

As regards the immunohistochemical analysis, the incubation was performed overnight at 4 °C with the following primary antibodies: BMAL1 (1:300, Absin, China), matrix degradation enzymes matrix metalloproteinase-3 (MMP-3) (1:400, Servicebio, China), MMP-13 (1:500, Absin, China), a disintegrin and metalloproteinase with thrombospondin motifs 4 (ADAMTS-4) (1:500, Servicebio, China), Collagen II (1:300, Servicebio, China), and osteoprotegerin (OPG) (1:200, Servicebio, China). The tissues were treated with secondary antibodies (HRP labeled) of the corresponding species of the primary antibody and incubated. The slices were dehydrated, sealed, and examined under the microscope (NIKON ECLIPSE E100, Japan). As regards the immunofluorescence analysis, the incubation was performed overnight with the following primary antibodies, CD206 (1:500, Absin, China), Ki67 (1:500, Servicebio, China), F4/80 (1:500, Servicebio, China), receptor activator of nuclear factor-kappa B ligand (RANKL) (1:2000, Absin, China), and OPG (1:200, Servicebio, China). The tissues were treated with the secondary antibody and DAPI solution in the dark. Images were taken by fluorescent microscopy (NIKON ECLIPSE C1, Japan).

For IHC analysis, nucleus pulposus (NP) tissue from humans was fixed with 4% paraformaldehyde for 24 h and then processed via routine paraffin embedding, sectioning, and deparaffinization. For the caudal IVDs of mice, the tissue was harvested and fixed with 4% paraformaldehyde for 24 h, then decalcified using 10% ethylenediaminetetraacetic acid (EDTA) for 1 month before routine paraffin embedding, sectioning, and deparaffinization. The sections were stained by a routine H-E and Safranin O-Fast Green Staining method. Subsequently, the sections were incubated with a rabbit polyclonal antibody GPCR 35 (Cat No. PA5-23237, Thermo Fisher, USA), collagen II (Cat No. GB11021, Servicebio, China), or aggrecan (cat No. GB11373, Servicebio, China) at 4°C overnight. A specific IHC kit (cat. No. K5007, Agilent DAKO Inc., CA, USA) was used for the process according to the manufacturer's protocol. Nuclei were counterstained with hemalum (cat. G1004, Servicebio Inc., Wuhan, China). The stained samples were observed and photographed under a microscope (Axio, Carl Zeiss, Oberkochen, Germany).