Anti human cd31

Primary cultured fibroblasts were examined by immunofluorescence using anti-cytokeratin-18 (Sigma), human specific anti-vimentin (V9; Novocastra laboratories, Ltd., UK), anti-fibroblast surface protein (1B10; Sigma), anti-human CD31 (Santa Cruz, California), anti-α–SMA (1A4; Dako, Denmark), anti-PDGFRα (Dako), anti-human CD68 (1/100, BD Pharmingen™), anti-human CD163 (1/100, Bio-Rad), and anti-human F4/80 (1/50, Invitrogen) antibodies. The antibodies anti-AM, anti-CLR, anti-RAMP2, and anti-RAMP3 were developed and characterized in the laboratory.

Cells (total 2 × 10⁶) were suspended in 200 µL of ice-cold phenol red-free Matrigel (BD Bioscience, Franklin Lakes, NJ, USA) at ratios of 100:0, 50:50, and 0:100 (HUVECs:ahMNCs). The mixtures were then transplanted subcutaneously into the dorsal surface of Balb-c/nu mice (6-week-old, male) (Orient Bio, Seongnam, Korea) in accordance with previous reports [23 (link),47 (link),48 (link)]. At 4 days after injection, the Matrigel plugs were recovered, fixed with 10% buffered formalin, and then embedded in paraffin to prepare 4-µm-thick sections. For histological analysis, H&E staining was applied. For immunofluorescence, deparaffinized and rehydrated sections were incubated in target retrieval solution (Dako, Carpentaria, CA, USA) at 125 °C for 30 min. Anti-human CD31 (Santa Cruz Biotechnology) and anti-alpha smooth muscle actin (αSMA) (Dako) antibody were applied to slides at 4 °C overnight. Nuclei were counter-stained by DAPI (1:1000, ThermoFischer Scientific, Carlsbad, CA, USA) for 5 mins at RT.