F4 80 clone ci a3 1

To detect infiltrated immune cells, paw sections (3.5 µm) were fixed with acetone and denatured with 100, 95, and 75% ethanol for 15 s separately. For antigen retrieval, sections were heated in Tris-EDTA retrieval buffer (10 mM Tris Base, 1 mM EDTA solution, 0.05% Tween 20, pH 9.0) in a water bath at 98°C for 20 min. After cooling down at RT for 30 min, sections were incubated with antibodies against F4/80 (clone CI:A3-1; Abcam), GR-1 (clone RB6-8C5; R&D), CD4 (clone 4SM95; Thermo Fisher), CD8 (clone 4SM15; Thermo Fisher), and CD19 (clone 6OMP31; Thermo Fisher) at 4°C overnight for detecting macrophages, neutrophils, CD4+ T cells, CD8+ T cells, and B cells respectively. Next, sections were washed with PBS and incubated with anti-Rat IgG (Alexa Fluor 488 conjugated; Thermo Fisher) secondary antibody. Cell nucleus was counterstained with Hoechst 33342 (Thermo Fisher), and sections were visualized using a fluorescence microscope (Olympus BX61). Quantification of fluorescence intensity was performed by count function in the software of CellSens dimension (Olympus). Briefly, five different regions in arthritis paw from DTHA mice and normal paw from untreated mice with strongest fluorescence intensity were selected for each sample. Then the average value of fluorescence intensity was calculated from these five regions and used for the next statistical analysis.