Rat monoclonal anti f4 80 clone a3 1

Primary stromal vascular fractions (SVF) from ingSAT, pgVAT and iBAT were prepared as described^{22 ,23} . Cells were washed in phosphate-buffered saline (PBS) supplemented with 0.1% BSA and 0.5 mM EDTA, pH 8.0, and stained when appropriate with rat monoclonal anti-F4/80 (Clone A3-1, cat # ab105155, 1:200, Abcam), rat monoclonal anti-CD11b (Clone M1/70, cat # 561114, 1:200, BD Biosciences) and rat monoclonal anti-CD301 (Clone LOM-14, cat # 145705, 1:400, BioLegend) for macrophage staining, or rat monoclonal anti-CD45 (Clone 104, cat #558702, 1:200, BD Biosciences) and rat monoclonal anti-Siglec F (Clone E50-2440, cat # 562068, 1:200, BD Biosciences) for eosinophil staining. For intracellular markers, cells were subsequently washed again, fixed in 2% paraformaldehyde, then labeled with rat monoclonal anti-NOS2 (Clone CXNFT, cat # 61-5920, eBioscience) and rabbit monoclonal anti-TH (Clone EP1533Y, cat # TA303716, 1:50, Origene) and polyclonal goat anti-rabbit secondary antibody (cat # A-10931, 1:400, Thermo Fisher Scientific) in permeabilization buffer (PBS with 0.5% Tween 20). Data were acquired using a Gallios Flow Cytometer (Beckman Coulter) and analyzed with FlowJo v10 software. After gating out dead cells and doublets, macrophages were identified as CD11b⁺F4/80⁺ and eosinophils were detected as CD45⁺Siglec-F⁺ cells.