Anti mouse f4 80 fitc

Immediately after sacrifice, bone marrow cells were flushed with FACS buffer (PBS supplemented with 2% FBS and 2 mM EDTA) and 1 × 10⁶ cells were incubated with fluorescent-conjugated antibodies. Antibodies for flow cytometric analyses included: anti-mouse F4/80 FITC (Abcam, Clone A3-1), F4/80 APC (Biolegend, Clone A3-1), anti-mouse CD11B APC (eBioscience, Clone M1/70), CD206 PE (AbD Serotec, Clone MR5D3), anti-mouse GR-1 PEcy3 (BDBiosciences, Clone RB6-8C5) and CD86 PE (BDBiosciences, Clone GL1). C-FMS+ cells were identified by GFP expressed in the MAFIA mice. Matched isotype controls were used and pre-gating was performed excluding dead cells and debris in SCC x FSC graph and aggregates in FSC-W x FSC-H and SSC-W x SSC-H.
For macrophage analysis in tumor tissue, tumors were mechanically dissociated, followed by digestion in complete RPMI-1640 media supplemented with type I collagenase (5 mg/mL; Sigma-Aldrich), 0.5% FBS and 1% Penicillin-streptomycin for 4 hours. Cells were washed in PBS multiple times to eliminate collagenase and passed through 75μm cell strainer for single cell suspension. Viable cells were counted and 1 × 10⁶ cells incubated in FACs staining buffer containing combinations of antibodies or isotype controls. After washing, cells were analyzed on BD FACSAria™ III.

For immunofluorescence analysis, frozen sections of liver graft specimens were incubated with anti-mouse F4/80-FITC (Abcam, Cambridge, MA, USA) at 4 °C overnight.
Proliferating Kupffer cells in frozen graft sections were stained with anti-mouse F4/80-Alexa Flour 647 (Abcam) and anti-Ki67 antibodies (Abcam) at 4 °C overnight. Sections were then incubated with a goat-anti-rat IgG-Alexa Flour 488 secondary antibody (Abcam) for 2 h at room temperature.
Apoptotic Kupffer cells in frozen graft sections were stained with anti-mouse F4/80-Alexa Flour 647 (Abcam) and detected in a TUNEL assay with an ApopTag® Plus Peroxidase in Situ Apoptosis Kit (EMD Millipore, Darmstadt, Germany). Detection and all measurements were carried out following the manufacturer’s instructions.
All of the sections were then mounted in Mounting Medium with DAPI (Vector Laboratories, Burlingame, CA, USA) and scanned with a two-photon laser confocal microscope (Olympus, Tokyo, Japan).