Aggrecan

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).

The IVD tissues were decalcified, embedded in paraffin, and cut into 5 m slices after being fixed in 4% paraformaldehyde. The paraffin slices were antigen-repaired with citric acid (pH 6.0), blocked with goat serum, and dewaxed with xylene and gradient ethanol. The sections were then treated at 4 °C for an overnight period with primary antibodies to ACSL4 (1:200, Proteintech, USA), Collagen II (1:200, Novus, USA), Aggrecan (1:500, Servicebio, China), and Piezo1 (1:200, Affinity, USA). The sections were exposed to goat anti-rabbit IgG-HRP secondary antibody for 1 h at room temperature the next day. The DAB Substrate kit (ZLI-9018, ZSGB) was used for detection, and the samples were then counterstained with 1% hematoxylin. Using a microscope (Leica DMI3000B, Germany), pictures were recorded. ImageJ quantified the areas that were positive.

Tissues were fixed in 4% paraformaldehyde for 24 h and then decalcified in 10% EDTA (pH 7.4) for 30 days. After paraffin embedding, tissues were cut into 5‐μm‐thick sections. Medial and lateral intervals were used for tissue sectioning with a spacing of 200 μm. After deparaffinization in xylene, the sections were rehydrated using a graded ethanol series. Then, they were stained with Safranin O/Fast Green (Solarbio; G1371‐5), haematoxylin and eosin, and toluidine blue. Immunohistochemical staining of Aggrecan (Servicebio, Wuhan, China; GB11373), COL‐I (Servicebio, Wuhan, China; GB11022), and COL‐II (Servicebio, Wuhan, China; GB11021) were then performed according to a previous protocol.
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