Fitc conjugated anti mouse antibody

BALB/cByJ mice were put in the hyperbaric chamber for 1.5 h in a hyperoxic atmosphere (75% O₂). Mice were sacrificed immediately through CO₂ narcosis, followed by the rapid exposure of the abdominal cavity and cannulation of the hepatic portal vein using a 26-gauge needle. After that, the inferior vena cava was immediately incisioned to enable drainage. 10mL of PBS 1X per mouse were perfused in total. Then, the livers were collected in Petri dishes with PBS 1X at 3% of FBS. The livers were cut into small pieces with scissors and put in 1mL of collagenase solution (0,5 mg of collagenase type IV/1 ml of RPMI 1640 medium with Type IV DNase I; final concentration: 25units/ml, Sigma-Aldrich), and incubated for 10 min at 37 °C. After stopping the reaction and passing the liver pieces through a strainer, the cells were washed and centrifuged at 50 g or 200 rpm for 3 min to discard the hepatocytes. The supernatant was centrifuged and osmotic lysis buffer of RBCs (ammonium chloride/potassium hydrogen carbonate buffer) was added over the pellet. After washing, the cells were stained with F4/80 (using FITC-conjugated anti-mouse antibody, Biolegend) and SIRP-α (PE-conjugated anti-mouse antibody, Biolegend). Both antibodies were used at 2 μg/μl.