Apc conjugated anti mouse cd11b

c-Fms tyrosine kinase inhibitor, Ki20227 (N-[16 (link)]-N′-[1-(1,3-thiazole-2-yl)ethyl]urea) was purchased from AdooQ (Irvine, Cat^#A11472-50). Mouse RANKL was purchased from Novoprotein (Cat^#CR06). Reduced Matrigel was purchased from Corning (Cat^#356230).PE-conjugated anti-mouse podoplanin (PDPN, Cat^#127408), violet 605-conjugated anti-mouse CD115 (=c-Fms, Cat^#135517), and Cy5.5-conjugated anti-mouse CD105 (Cat^#120415) antibodies were purchased from Biolegend. Biotin-conjugated anti-mouse Lymphatic Vessel Endothelial Hyaluronan Receptor 1(LYVE-1, Cat^#13-0443-80), FITC-conjugated anti-mouse CD31 (Cat^#11-0311-81), APC-conjugated anti-mouse CD11b (Cat^#17-0112-83) antibodies were purchased from eBioscience. Streptavidin-conjugated APC was purchased from BD Pharmingen (Cat^#554067). Hamster anti-mouse PDPN (Abcam, Cat^#ab11936), FITC-conjugated anti-mouse smooth muscle actin (Sigma, Cat^#F3777), goat anti-mouse VEGFR3 (Cat^#AF743) antibodies were purchased from R&D Systems. Anti-mouse Prox1 antibody (Cat^#11-002) was purchased from AngioBio. Alexa flour 488-conjugated anti-goat (Cat^#A11078) and Alexa flour 568-conjugated anti-hamster (Cat^#A21111) antibodies were purchased from Invitrogen.

Epididymal fat pads were isolated after saline perfusion, then minced, washed, and centrifuged (1,000 g for 5 min). Floating adipose tissue was collected and digested with type I collagenase solution (1 mg/ml, 30 min). This digest was pelleted to yield the SVF, which was washed twice in FACS wash buffer (PBS supplemented with 4% FBS), then blocked for Fc receptors using anti-mouse CD16/32 (BD Bioscience), followed by staining with APC-conjugated anti-mouse CD11b, eFluor 450- conjugated anti-mouse CD11c, and PE-conjugated anti-mouse F4/80 (eBioscience). Other surface markers included CD18 and ICAM-1 (both PE-conjugated, eBioscience). Samples were analyzed using a BD LSD II flow cytometer.