Eclipse e600 microscope

Cells used for immunofluorescence were fixed for 30 minutes at room temperature in 4%PFA, while slides were incubated for 5 minutes in PBS to remove residual OCT. After washing with PBS, samples were blocked in serum of the host of the secondary antibody (5% serum and 0.3% BSA in PBS with 0.2% Triton-X 100), and then incubated overnight with rabbit anti-mouse Iba1 (1:500, Wako), mouse anti-mouse iNOS (1:500, BD Biosciences), mouse anti-mouse Arg-1 (1:500, BD Biosciences), rat anti-mouse CD206 (1:50, R&D Systems), rat anti-mouse CD86 (1:50, Millipore), rat anti-mouse CD11b (1:200, Serotec), and rabbit anti-mouse p-Stat6 (1:100, Cell Signaling) in 0.3% BSA in PBS with 0.2% Triton-X 100. After washing with PBS, sections were incubated with fluorescence-conjugated FITC or Cy3 goat anti-rabbit or rabbit anti-mouse secondary antibody and Streptavidin-conjugated Cy3 (to detect bound biotinylated tuftsin) for 1 hour at room temperature, washed 3 times with PBS, and mounted using Fluoromount-G with DAPI (Southern Biotech, USA). For experiments where two markers were used for staining, (e.g. Iba1/iNOS or Iba1/Arg1), yellow fluorescence is indicative of double-positive signal. DAPI was included in images as an indicator of cell density in lesion areas. The cells were imaged using a Nikon Eclipse E600 microscope or a Zeiss LSM 510 confocal microscope.

Spinal cord sections mounted on slides for immunofluorescence were rinsed in PBS for 5 min to remove residual OCT. After washing, samples were blocked in serum of the host of the secondary antibody (5% serum and 0.3% BSA in PBS with 0.2% Triton-X 100) and then incubated overnight with rabbit anti-mouse Iba1 (1:500, Wako), mouse anti-mouse iNOS (1:500, BD Biosciences), mouse anti-mouse Arg-1 (1:500, BD Biosciences), rat anti-mouse CD86 (1:500, BD Biosciences), goat anti-mouse CD206 (1:100, R&D Systems), rabbit anti-mouse GFAP (1:1000, Dako) rabbit anti-mouse NG2 (1:500, a generous gift from the Levine lab), mouse anti-mouse CC1 (1:100, EMD Millipore), rat anti-mouse MBP (1:50, Bio-Rad), or rabbit anti-mouse neurofilament-L (1:100, EMD Millipore) in 0.3% BSA in PBS with 0.2% Triton-X 100. After washing with PBS, sections were incubated with fluorescence-conjugated FITC or Cy3 goat anti-rabbit, goat anti-mouse, goat anti-rat, or donkey anti-goat antibody for 1 h at room temperature, washed three times with PBS, and mounted using Fluoromount-G with DAPI (Southern Biotech, USA). The sections were imaged using a Nikon Eclipse E600 microscope and Zeiss LSM 510 confocal microscope.

After completion of SPECT and MRI imaging, animals were euthanized. The joint capsules from 4 animals per group were collected and were decalcified. Additionally, the synovial membranes from rats were surgically excised. They were then paraffin-embedded and routinely processed to produce 5 μm micro-sections. The tissue sections were stained by Safranin O/Fast green and Hematoxylin/Eosin, respectively. Microscopic images of each section were taken by a Nikon ECLIPSE E600 microscope with a Zeiss software.

Cells were grown on coverslips coated with poly-lysine until 75% confluent. The cells were then washed twice in ice-cold phosphate buffered saline (PBS), fixed with 4% paraformaldehyde for 20 min at room temperature, permeabilized with 0.5% Triton in 1% glycine and then blocked using 0.5% BSA and 5% goat serum for 30 min at room temperature. Samples were then incubated with antibodies against P63 (Thermo Scientific, catalog # 703809, 1:200 dilution), and cytokeratin 14 (Santa Cruz Biotechnology, catalog #sc-53253, 1:200 dilution) in blocking solution overnight at 4 °C. Samples were then washed three times with PBS containing Tween 20 (PBST), followed by incubation with FITC-and Alexa 568- conjugated secondary antibodies (Invitrogen, 1:1000 dilution). Nuclei were also stained with 4', 6-diamidino-2-phenylindole (DAPI) (Invitrogen, 1:5000 dilution) before cells were mounted. Cells were observed using a Nikon Eclipse E600 microscope and a Zeiss confocal laser-scanning microscope.