A3107

Chondrocytes grown on the 6-well Flexcell culture plates were transferred to glass coverslips. After adhesion, chondrocytes were rinsed with PBS and fixed with 4% paraformaldehyde at room temperature for 15 min, then rinsed with PBS. Then, 1% Triton X-100 and 5% normal goat serum were added for permeabilizing for 15 min and binding nonspecific protein for 1 h, respectively. After that, chondrocytes were incubated with anti-Piezo1 (1:200, DF12083, Affinity, Liyang, China), anti-NFAT1 (1:200, A3107, Abclonal, Wuhan, China), anti-MMP3 (1:100, A1202; Abclonal, Wuhan, China), anti-MMP13 (1:200, GB11247; Servicebio, Wuhan, China), anti-COL2A1 (1:200, TA0135; Abmart, Shanghai, China), and anti-Aggrecan (1:200, TD7561; Abmart, Shanghai, China) antibodies overnight at 4 °C. After rinsing with PBST, chondrocytes were incubated with anti-mouse or anti-rabbit conjugated with CY3 and FITC for 1 h at room temperature. Next, chondrocytes were rinsed with PBST and stained with DAPI. Images were captured randomly on an Olympus BX53 fluorescence microscope. ImageJ software (version: 1.8.0) was used to quantify the mean immunofluorescent intensity.

Tissue sections were prepared as described in the previous section. After deparaffinization and rehydration through ethanol gradients (95%, 80%, 70%), sections underwent an antigen repair process in sodium citrate buffer using microwave thermal repair and endogenous peroxidase blocking in 0.3% hydrogen peroxide. After nonspecific reactions were blocked with 5% goat serum at 20℃ for 10 min. Sections were first incubated with anti-COLIII primary antibody (GB111629, Servicebio, Wuhan China) overnight at 4 °C, then incubated with horseradish peroxidase-labeled goat anti-rabbit secondary antibody, and fluorescent dye was ligated to it according to the manufacturer’s instructions (abs50012, Absin, Shanghai, China). After washing with PBST 3 times, sections were further incubated with the second primary antibody (anti-SOX5 (A6985, Abclonal, Wuhan, China), anti-NFATC2 (A3107, Abclonal), anti-NPAS2 (A16930, Abclonal)) at room temperature for 1 h. The procedures were repeated starting from the second antibody incubation. After all the procedures were completed, the slides were washed in PBST, counterstained with DAPI, and sealed with anti-fluorescence quenching sealing tablets. The stained tissue sections were examined using a Leica DMI8 microscope (Leica, Germany).

After 7 days of SCI, the rats were perfused with paraformaldehyde, dissected, and sectioned at the site of injury, paraformaldehyde-fixed, sucrose sunk and OCT embedded, followed by sectioning. Sections were treated with 0.3% TritonX-100 for 20 min and blocked with 5% BSA blocking solution at room temperature for 1 h. The appropriate primary antibody was added to the sections and incubated overnight at 4°C, including anti-FECR1G (1:100, DF13263, Affbiotech, China), anti-NFATC2 (1:100, A3107, Abclonal, China), and anti-iNOS (1:100, ab210823, Abcam, United Kingdom). Sections were incubated with a secondary antibody (1:200) at room temperature in the dark for 1 h. After washing with PBS for 10 min, cell nuclei were stained with DAPI.