Ar0030

Immunofluorescence staining of paraffin-embedded human liver tissue sections was performed using anti-PD-1/CD279 (66220-1-Ig, Proteintech, Rosemont, IL, United States) and anti-CD8α (PB9249, Boster Bio, Pleasanton, CA, United States) antibodies. Antigen retrieval was performed with EDTA buffer (AR0023, Boster Bio). Tissue sections were blocked with 10% goat serum (AR1009, Boster Bio), and then incubated with rabbit anti-CD8α antibody (1:200 dilution) and mouse anti-PD-1 antibody (1:4000 dilution) at 4 °C overnight. Secondary antibodies including DyLight594 fluorescein goat anti-mouse (BA1141, Boster Bio) and fluorescein DyLight488 goat anti-rabbit (BA1127, Boster Bio) IgGs were then added and incubated for 45 min at 37 °C. DAPI staining solution (AR1176, Boster Bio) was used for counterstaining at room temperature for 3 min, and then washed with PBS (pH 7.2-7.6) (AR0030, Boster Bio). Slides were mounted using anti-fluorescent quench mounting medium (AR1109, Boster Bio). Finally, whole-slide scanning (APERIOVERSA8, Leica, Wetzlar, Germany) and image acquisition (BX51, Olympus, Tokyo, Japan) were performed.

CoCrMo particles, a gift from Dr. Zhenzhong Zhang (The College of Materials Science and Engineering of Nanjing University of Technology), had a mean diameter of 51.7 nm. For decontamination from endotoxins, the particles were autoclaved for 15 min at 121°C and 15 psi. This treatment resulted in negative testing for endotoxin using a quantitative Limulus Amebocyte Lysate (LAL) Assay (Charles River, R13025) at a detection level of <0.25% EU/ml. The particles were suspended in phosphate-buffered saline (PBS; Boster Biological Technology Co., Ltd., AR0030, pH 7.2 to 7.4) at a concentration of 50 mg/ml as stock solutions. For in vitro experiments, the particles were further diluted in cell culture medium to attain concentrations ranging from 10 to 200 µg/ml and ultrasonicated for 20 min before exposing them to cells.