Ab219620

Primary mouse chondrocytes were rinsed in PBS and fixed with 4% paraformaldehyde for 15 min at room temperature. After being permeabilized with 0.3% Triton X-100 for 5 min, the cells were blocked with 1% BSA for 30 min and incubated with mouse anti-MMP-13 (1:300, Abcam, ab219620) and anti-Col2a1 (1:500, Abcam, ab34712) antibodies at 4 °C overnight. The samples were then washed with PBS (4 °C) three times and incubated with a Cy3-conjugated goat anti-rabbit secondary antibody (1:50, ASPEN, AS1109) at 37 °C for 30 min. TUNEL staining of chondrocytes was performed with a TUNEL Assay Kit (Roche, Basel, Switzerland) according to the manufacturer’s protocol.

Paraffin-embedded NP tissue slices (4 μm thickness) were placed in ethylenediaminetetraacetic acid (EDTA) buffer solution (0.05 mol/L Tris + 0.001 mol/L EDTA; pH 8.5) and heated for 21 min in a microwave oven for antigen retrieval. The tissue slices were incubated in 0.3% H₂O₂ for 10 min, followed by blocking using 5% bovine serum albumin (BSA) for 20 min. The tissue slices were probed overnight at 4°C with the following primary antibodies: rabbit anti-MMP13 (ab219620, 1: 500, Abcam, Cambridge, UK), rabbit anti-ADAMTS 5 (PA5-27165, 1 : 100, Invitrogen, Carlsbad, CA), rabbit anti-COL II (15943-1-AP, 1 : 200, Proteintech Group Inc., Chicago, IL), rabbit anti-aggrecan (13880-1-AP, 1 : 200, Proteintech), and rabbit anti-GPX4 (ab125066, 1 : 100, Abcam). Then, the slices were further incubated with biotinylated secondary antibody goat anti-rabbit IgG (ab205718, 1 : 2500, Abcam) for 20 min. The slices were washed with 0.1 M PBS and reprobed with streptomycin albumin working solution labeled with horseradish peroxidase for 20 min. Next, the slices were colored with diaminobenzidine, counterstained with hematoxylin, and observed under a microscope (Leica-DM2500, Leica, Wetzlar, Germany). Staining quantification was performed using ImagePro Plus 7.1 software (Media Cybernetics, Silver Spring, MD).