Immunofluorescence labelling of ProtheraCytes induced to endothelial differentiation
ADHESION, FLUOR
ProtheraCytes immobilized on slides after cytospin centrifugation and ProtheraCytes undergoing endothelial differentiation in chamber slides were fixed in methanol for 10 min at 4 °C and blocked in 5% BSA for 1 h at room temperature before antibodies were added. Primary antibodies were specific for CD31 (Ozyme), von Willebrand factor (vWF, Agilent Technologies, France), Vascular Endothelial Growth Factor Receptor 2 (VEGFR-2, Miltenyi Biotech France), and Vascular cell adhesion protein 1 (VCAM-1, Abcam, Netherlands). Secondary antibodies used were goat anti-Rabbit Alexa Fluor 488 (Fischer Scientific, France), goat anti-mouse Alexa Fluor 488 (Fisher Scientific, France) and goat anti-mouse Alexa Fluor 555 (Fisher Scientific, France). DAPI (CliniSciences, France) was used to visualize nuclei. Staining was analyzed by fluorescence microscopy.