Mouse anti mouse inos

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.