Vs200 system

The knee joints were obtained from the OA and processed with safranin-O and fast green (S&F) and hematoxylin & eosin (H&E). For the OA model, the severity of synovitis was assessed by the synovitis scores and the severity of cartilage damage was calculated by Osteoarthritis Research Society International (OARSI) scoring system [40 (link)]. Immunohistochemical staining was accomplished with the knee joint cartilages. The primary antibodies used in the experiments were those against matrix metallopeptidase 13 (MMP13) (ABclonal), A disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5) (ABclonal), Collagen II (ProteinTech), Aggrecan (ProteinTech), Cleaved Caspase-3 (Cell Signaling Technology), PIM-1(HUABIO). The images of immunohistochemical staining were captured using an Olympus VS200 system (Tokyo, Japan).

Oil Red O staining was performed according to Yang et al. and the manufacturer’s instructions (Yang et al., 2023 ). The working solution of Oil Red O (Sigma–Aldrich, #O1391) was prepared by diluting it from the stock solution, mixing it with water in a 3:2 ratio, and then filtering it through 0.45 μm filters to eliminate any impurities. The OCT-embedded synovial sections were loaded with Oil Red O working solution for 15 min at RT. Slides were washed three times in water without shaking, sections were then counterstained with hematoxylin for 5 min. Images were obtained using Olympus VS200 system, and area of lipid droplets was quantified using the ImageJ software.

The paraffin-embedded synovial sections were deparaffinized with xylene, followed by rehydration through a graded ethanol series and rinsing with running tap water. Masson-trichrome staining was detected based on the manufacturers’ instruction (Solarbio, G1340). Images were collected by Olympus VS200 system.

Paraffin sections were deparaffinized and rehydrated, and antigen was retrieved by microwave heat treatment in citrate buffer (pH 6.0) for 25 min and then penetrated with 0.3% Triton X-100 in 1× PBS for 1 h, and afterward placed in 5% blocking buffer (normal donkey serum, 017-000-021; Jackson) for 1 h at RT. Primary antibodies were incubated in a humidified chamber overnight at 4°C. Then slides were incubated with 3% H₂O₂ for 15 min for the inactivation of endogenous peroxidase before incubation with a secondary antibody, followed by colorimetric detection using DAB kit (ZSGB-BIO) and then counterstaining with hematoxylin for 5 min. Finally, the sections were dehydrated and mounted in neutral resinous mounting medium. Images were captured by using an Olympus VS200 system. The following antibodies were used for Immunohistochemistry: anti-CD31 (R&D Systems, AF3628), anti-P21^Cip1 (Cell Signaling Technology, 2947), anti-ERVK7 (United States Biological, 302427), anti-H3K9me3 (Abcam, ab8898), anti-S100A8 (Abcam, ab180735), anti-S100A9 (Abcam, ab92507), anti-FOXO1 (Cell Signaling Technology, 2880), and anti-VCAM1 (Abcam, ab134047).