Macrophage immunofluorescence

After fixation, cells were washed three times for 5 minutes each with 1X PBS and stored in 1X PBS at 4C until ready for immunofluorescence staining. Cells were blocked in donkey blocking buffer (5% donkey serum (Fisher Scientific), 1% bovine serum albumin (Sigma), 0.1% Tween-20-Triton X-100 (Fisher Scientific)) for 1 hour at room temperature. Following block, cells were stained with primary antibody, either rabbit anti-iNOS (ab3523, Abcam, Cambridge, UK) diluted 1:100 in donkey blocking buffer or goat anti-liver arginase (ab91279, Abcam) diluted 1:50 in donkey blocking buffer, for 14-16 hours in the dark at 4° C. Cells were then washed three times with 1X PBS for five minutes per wash. Cells incubated with rabbit anti-iNOS primary were incubated with donkey anti-rabbit Alexa Fluor 594 secondary antibody (ab150064, Abcam) diluted 1:200 in donkey blocking buffer. Cell incubated with goat anti-liver arginase were incubated with donkey anti-goat Alexa Fluor 594 (ab150136, abcam) diluted 1:200 in donkey blocking buffer. Cells were incubated with secondary antibody for 1 hour at room temperature in the dark. Cells were washed three times with 1X PBS for five minutes per wash. Cells were then incubated with 4′,6-diamidino-2-phenylindole (DAPI) nuclear stain (Biolegend 422801, San Diego, CA) for 1 minute in the dark at room temperature. Cells were washed five times for 5 minutes per wash in 1X PBS. Cells were stored in PBS in the dark at 4° C until ready for imaging. Immunofluorescence images were captured using a FLoid cell imaging station (Thermo Scientific, Waltham, MA). Acquisition settings were set at: blue laser intensity=30%, red laser intensity=50%, optical zoom=0%.