Immunofluorescence staining

For immunofluorescence imaging, cells were grown to 70% confluence on 8-well glass culture slides (Merck Millipore, Tullagreen, Carrigtwohill, Ireland), washed, fixed with 2% formaldehyde, and washed again in PBS. Primary antibodies were added at 1:100 dilutions and slides were incubated at 4° on a rocker overnight. Cells were then washed thoroughly with PBS and secondary antibodies were added at a 1:100 dilution for 1 hour. After washing with PBS, slides were cover-slipped using ProLong Diamond Antifade Mountant with DAPI (Invitrogen, Eugene, OR) and glass coverslips. Primary antibodies were rat αCD31, αCD140a, and αCD324 (Beckton Dickinson) and isotype control. The secondary antibody used was chicken anti-rat IgG AF488 (Invitrogen).