Fitc labeled anti mouse ly 6g

For (immuno)histological examination, 4μm paraffin sections were cut. Tubular injury was determined on sections stained with periodic acid Schiff after diastase (PasD). The percentage of damaged tubules in the corticomedullary region and in the outer cortex was estimated by a pathologist blinded for the groups using a 5-point scale according to the presence of necrosis in 10 randomly chosen non-overlapping high power fields (hpf, magnification 400x). Injury was graded as follows: 0 = 0%, 1 = <10%, 2 = 10–25%, 3 = 25–50%, 4 = 50–75%, and 5 = >75%. Immunohistochemical stainings to detect macrophages, neutrophils, apoptosis and proliferation were performed using rat-anti-mouse F4/80 (Serotec, Oxford, UK), FITC-labeled anti-mouse Ly-6G (BD Biosciences), rabbit-anti-mouse active caspase 3 (Cell Signaling Technology, Beverly, MA, USA) and rabbit-anti-human Ki67 (clone Sp6; Lab Vision Corp.) respectively. Macrophages and apoptotic and proliferating TEC were counted in the cortico-medullar region in 10 randomly chosen, non-overlapping hpf (magnification 400x). Total number of neutrophils was counted in one kidney sections per mouse.

BALF was centrifuged at 250 g for 10 min, after which the supernatant was collected and stored at −20°C for cytokine analyses. The cell pellets were labeled with FITC-labeled anti-mouse Ly-6G, PE-labeled anti-mouse CD3, PE-Fluor610-labeled anti-mouse CD45, PerCP-Cy5.5-labeled anti-mouse CD11c, PE-Cy7-labeled anti-mouse CD11b, AlexaFluor647-labeled anti-mouse Siglec F, AlexaFluor700-labeled anti-mouse Ly-6C (all from BD Biosciences), and efluor780-labeled fixable viability dye (eBioscience). After 30 min of staining, 1-step Fix/Lyse Solution (eBioscience/Thermo Fisher) was added. After 60 min, the cells were washed and resuspended in FACS buffer supplemented with 7000 Precision Counting Beads (BioLegend). The gating strategy is presented in Fig. S1.