Anti mouse cd11b m1 70

A portion of the lungs was weighed, incubated in 2 mg ml⁻¹ collagenase D (Roche) and 40 U ml⁻¹ DNase I (Roche) and dispersed by passage through 70 mm mesh. After red blood cell lysis, viable cells were counted and incubated with fluorescence-conjugated antibodies for labeling. The following specific FACS antibodies (BD Pharmingen, San Diego, CA, USA) were used: Gr-1 (RB6-8C5), CD11b (M1/70), Ly6G (1A8), CD3e (145-2C11) and CD19 (1D3). To determine CD11b and CD18 expression on neutrophils, purified neutrophils were incubated with anti-mouse CD11b (M1/70) and CD18 (C71/16) (BD Pharmingen). After incubation, cells were analyzed on a FACS Calibur instrument (BD Biosciences). The isotype control antibodies used in this experiment were obtained from BD Pharmingen.

Eyes were frozen in OCT medium (Fisher Scientific, UK) and 12-μm cryosections stained with anti-mouse Ly6C-PE (HK1.4, eBioscience), anti-mouse Ly6G-APC (1A8, BioLegend), anti-mouse CD11b (M1/70, BD Biosciences) and anti-CD45 (30-F11, eBioscience). Retinal flat-mounts were dissected and placed into 1% BSA/3% Triton X-100/ 5% normal goat serum and incubated overnight at 4°C with 1:200 anti-mouse Iba-1 (Wako, Osaka, Japan). Retinas were incubated with Alexa-Fluor-594 (Invitrogen Molecular Probes) secondary antibody for 4 h, mounted and imaged with a Leica DM5500Q confocal microscope.

LPS (Lot. No. L8274) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Anti-mouse CD11b (M1/70) and MHC Class I (H2K^d, SF1-1.1) monoclonal (m) Abs were purchased from BD Biosciences (San Jose, CA, USA). Anti-mouse CD16/CD32 (93), and Gr-1 (RB6-8C5) mAbs were purchased from eBioscience (San Diego, CA, USA). Anti-CD11c (N418), CD80 (16-10A1), MHC Class II (I-A/I-E, M5/114.15.2) and 7-amino-actinomycin D (7AAD) from Tonbo Biosciences (San Diego, CA, USA). Rabbit anti-MRC1 polyclonal (p) Ab was from Bioss Antibodies (Woburn, MA, USA). Alexa 568-conjugated goat anti-rabbit IgG was from Molecular Probes (Eugene, OR, USA).