Rabbit anti human cd146

Cells were seeded in eight-chamber slides, grown until confluence and then fixed with 4% wt/vol. paraformaldehyde for 15 min at room temperature. This step was followed by an incubation overnight at 4°C with the primary antibodies reported in ESM Table 3: rabbit anti-human CD146 (Abcam), rabbit anti-human CXCL12 (Cell Technologies, Cambridge, UK), rabbit anti-human vasclular endothelial-cadherin (VE-cadherin, Abcam), mouse anti-human nestin (Abcam), rabbit anti-human PDGFRβ (Santa Cruz, Heidelberg, Germany), rabbit-anti-human vascular endothelial growth factor receptor 2 (VEGFR2, Abcam), rabbit LEPROT (Abcam), and mouse VWF (Abcam). All incubations were performed overnight at 4°C in PBS. Then, the cells were washed with PBS and incubated with appropriate secondary antibody diluted 1:200 in PBS for 2 h at room temperature. Finally, cells were washed with PBS and counterstained with DAPI 1 μg/μl, for 1 min at room temperature. Images of random fields were obtained at ×200 magnification. Antibodies were validated previously by using negative and/or positive cell cultures as controls, according to the literature. Negative controls have been performed for each immunohistochemistry and immunocytochemistry experiment omitting the primary antibody.