Anti cd68

The synovial tissues were taken from normal and CIA rats and cut and digested with DNase (0.15 mg/mL), hyaluronidase (0.15 mg/mL), and collagenase (1 mg/mL). The normal FLS (normal FLS) and RA-FLS were obtained and resuspended with DMEM medium (Gibco, Gaithersburg, MD, USA) containing 10% fetal bovine serum (FBS, Tianhang Biotechnology Co., Ltd, Hangzhou, China), 30 mg/mL glutamine (Solarbio, Shanghai, China), 2.5 μg/mL amphotericin B (Sigma-Aldrich, St. Louis, Mo, USA), and 10 μg/mL gentamicin (Gibco). After the overnight culture, the nonadherent cells were removed, and adherent cells were routinely cultured.
The RA-FLS were identified by immunohistochemistry. In brief, the cells (2 × 10⁴ cells/mL) were cultured in 12-well plate for 48 h. Subsequently, cells were fixed with 4% paraformaldehyde and incubated with 0.1% TritonX-100 and 3% hydrogen peroxide. After washed, anti-Vimentin (1: 200, Wanleibio, Shenyang, China) and anti-CD68 (1: 200, Affinity Biosciences, OH, USA) were added and incubated overnight at 4°C. Following, horseradish peroxidase- (HRP-) labeled goat anti-rabbit IgG was added and incubated overnight at 37°C for 60 min. Finally, the cells were stained with diaminobenzidine (DAB) and hematoxylin. The images were captured by inverted microscope (BX53, Olympus, Tokyo, Japan).

We used the RIPA Lysis Buffer (Fudebio, Hangzhou, China) containing phosphatase/protease inhibitors (Fudebio, Hangzhou, China) to extract total protein from the cultured cells following the manufacturer’s method. SDS‐polyacrylamide gel electrophoresis (10%; SDS‐PAGE) was performed to separate the protein samples (20 µg), which were then transferred to Millipore polyvinylidene difluoride (PVDF) membranes. These PVDF membranes were kept in overnight incubation at 4°C with anti‐CD206, anti‐ODC, anti‐IL‐33, anti‐iNOS, anti‐CD68, anti‐ARG1 and anti‐GADPH primary antibodies (Affinity, USA), followed by incubation at room temperature for 60 minutes with horseradish peroxidase (HRP)–conjugated secondary antibodies (Affinity, USA) that were diluted to 1:5000 in 5% skim milk. After washing with TBST, the membranes were treated for 1 minutes with SuperSignal™ West Dura Extended Duration Substrate (Fudebio, Hangzhou, China) and were visualized through chemiluminescence.

For IHC staining, the sections were incubated with anti-cleaved-caspase3 (1:200; Affinity, China) or anti-p-NF-κB p65 (1:100; Affinity, China) antibodies and then incubated with the goat anti-rabbit secondary antibody (1:200; Affinity, China). The sections were then stained with DAB (Servicebio, China) and hematoxylin (Servicebio, China). For IF staining, the sections were incubated with anti-CD68 (1:50; Affinity, China) antibody, and the sections were incubated with a goat anti-rabbit IgG (H + L) cy3-conjugated secondary antibody (1:200; Affinity, China). Then, DAPI (Invitrogen, United States) staining was performed.