Immunohistofluorescence assays

To determine the expression of phospho-RSK2 and phopsho-FGFR3 in synovial tissues from RA and OA patients, the paraffin-invaded human synovial slices (5-μm) were deparaffinized by incubation at 60 °C for 2 h. The deparaffinized slides were rehydrated, unmasked by soaking in boiling 10 mM sodium citrate buffer (pH 6.0) for 10 min, and allowed to cool to room temperature gradually or treated with pepsin for 30 min. The slides were then blocked with 5% goat serum in 1 × PBX/0.5% Triton X-100 for 1 h at RT, and then hybridized with the indicated antibodies, phospho-FGFR3 (Y724) (1:50), phospho-RSK2 (T577) (1:50), CD68 (1:50) and CD4 (1:50), in 1 × PBS/0.5% Triton X-100 buffer overnight at 4 °C. The slides were washed and hybridized with secondary antibodies conjugated with Alexa-488 (Cat#: A11055, Bio-Rad Laboratories), −568 (Cat#: A11036, Bio-Rad Laboratories), or −647 (Cat#: A21235, Bio-Rad Laboratories) for 2 h at RT in the dark as indicated. Image stacks were captured using laser scanning confocal microscopy (LSM 710, Carl Zeiss Korea Co. Ltd., Seoul, Korea). To analyze the effects of kaempferol on signaling pathways for immune responsiveness in CIA-kaempferol treatment, the spleen tissue was snap-frozen in liquid nitrogen and stored at −70 °C. The spleen tissue sections (5-μm) were fixed in acetone and co-hybridized with a specific antibody using Fluorescein isothiocyanate-labeled anti-CD4 (Cat#: 553046, BD Biosciences, San Jose, CA, USA) and phycoerythrin (PE)-labeled anti-IL-17 (Cat#: 559502, BD Biosciences), PE-labeled anti-Src antibodies (Cat#: ab47405, Abcam, Cambridge, UK), PE-labeled anti-phospho-STAT3-Y705 (Cat#: 612569, BD Biosciences), or -S727 antibodies (Cat#: 558557, BD Biosciences). Image stacks were captured using laser scanning confocal microscopy (LSM 710, Carl Zeiss Korea Co. Ltd.). Positive cells were counted manually at a higher magnification (projected on a screen) by four individuals, and the results were expressed as means ± standard deviation.