Anti arginase 1 antibody

Lungs from mice (n>5 per condition, 15 weeks after radiation) were pulsed with 1 ml ice cold 5 mM EDTA instilled via intratracheal catheter. The lavage was repeated and pooled washings were centrifuged at 300 × g for 10 minutes. The cell pellet was resuspended in 2% BSA. After blocking the Fc receptor with purified anti-mouse CD16/CD32 antibody (#101335, BioLegend, San Diego, CA, USA), cells were labeled with a fluorophore-conjugated antibodies against F4/80 (#123116, BioLegend). Labeled cells were fixed with 2% PFA, permeabilized using a Permeabilization kit (FIX and PERM, Thermo Fisher, Waltham, MA, USA), and then labeled with an anti-Arginase 1 antibody (#89872, Cell Signaling Technology, Beverly, MA, USA) followed by an appropriate secondary antibody conjugated with a fluorophore (Thermo Fisher). At least 100,000 events were acquired for each sample with a CytoFlex (Beckman Coulter, Brea, CA, USA) and data were analyzed with FlowJo software (Tree Star, Inc., Ashland, OR, USA).

Peritoneal cells were cultured in a Millicell EZ Slide (Merck Millipore) for 48 h. Cells which grew in the slide were fixed, rinsed and stained using anti-Arginase-1 antibody and anti-Arginase-2 antibody, respectively (Cell Signaling Technology). After washing with PBS, the slides were incubated with a secondary antibody conjugated with fluorescein (Life Technologies, Carlsbad, CA, USA). For visualizing nuclei, the slides were mounted with DAPI (Cell Signaling Technology). Images were captured with a fluorescence microscope (Leica DM IRB, Germany).