Identifying Inflammatory Markers in Arthritis

Antigens were retrieved by incubating joint sections at 60 °C overnight with sodium citrate buffer (10 mM sodium citrate, 0.05% Tween 20, pH 6.0). The sections were blocked with 2% bovine serum albumin in phosphate-buffered saline (PBS), and then incubated with primary antibodies, including rabbit anti-BATF (Brookwood Biomedical), rabbit anti-RANKL (receptor activator of NF-κB ligand) (Abcam), goat anti-IL-6 (R&D Systems), rabbit anti-TNF-α (tumor necrosis factor alpha) (Novus Biologicals), and rabbit anti-Ki67 (Abcam). The Dako REAL Envision Detection system was used for chromogenic color development. BATF-expressing cells in synovial tissues were identified by double immunofluorescence labeling of vimentin for FLS, CD11b for macrophages, CD4 for T cells, and B220 for B cells. The following primary antibodies were used: mouse anti-CD4, mouse anti-B220, rat anti-CD11b (Abcam), rabbit anti-BATF (ThermoFisher Scientific), and mouse anti-vimentin (BD Pharmingen). Blood vessels in synovial tissues were detected with mouse anti-CD31 (Dianova). TRAP activity was determined in joint sections as previously described [20 (link), 22 (link)], and the numbers of TRAP-positive osteoclasts were counted in regions containing pannus-cartilage and pannus-bone interfaces.