Df6392

Cells and tissues were lysed by RIPA buffer containing protease and phosphatase inhibitors and PMSF. The proteins were separated by 10% SDS-PAGE and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Darmstadt, Germany). After blocking for 1.5 h in 5% skimmed milk diluted by 1 × PBS-T (0.5% Tween−20), the membranes were incubated overnight at 4°C with the following primary antibodies: anti-CX43 (ab11370, Abcam, MA, USA), anti-CD133 (abs131197, Absin, Shanghai, China), anti-CD44 (DF6392, Affinity Biosciences, OH, USA), anti-Nanog (AF5388, Affinity Biosciences), anti-SOX2 (ab92494, Abcam), anti-cleaved caspase-3 (AF7022, Affinity Biosciences), anti-cleaved caspase-9 (AF5240, Affinity Biosciences), anti-caspase-3 (AF6311, Affinity Biosciences) or anti-caspase-9 (AF6348, Affinity Biosciences). Anti-GAPDH (Proteintech Group, Wuhan, China) was used as protein-loading controls. Blots were incubated with HRP-conjugated secondary antibodies for 1 h at room temperature and visualized with ECL Western Blotting Substrate (ThermoFisher Scientific, IL, USA).

Tail veins of AIA rats were administered with free DiD or DiD-NPs. Ankle joints were collected 24 h after the last administration to prepare sections. The prepared sections of 10 μm thick slices were stained with CD44 antibody (Affinity Biosciences, DF6392, 1:500), CD68 antibody (Affinity Biosciences, DF7518, 1:500), and FOLR2 antibody (Affinity Biosciences, DF9518, 1:300). Nuclei was stained by DAPI. A laser scanning confocal microscope (LSM 800, Zeiss, Germany) was taken to record the fluorescent distributions in synovial joints.