Immunostaining of iPSC-derived Endothelial Cells

iPSCs were cultured in 6-well plates (Corning) and were differentiated into endothelial cells as described earlier. At day 3 cells were fixed in 4% paraformaldehyde (PFA) at room temperature for 20 min, permeabilized and immunostained following standard protocol using anti-Factor VIII antibody (Abcam, ab236284, 1:200) and fluorescently labeled secondary antibody (Invitrogen, Alexa fluor 546 goat anti-rabbit, 1:500). Additionally, nuclei were labeled with Hoechst 33342 (Invitrogen, H3570, 1:5000). Images of cells were automatically captured using an automated Operetta CLS confocal microscope (PerkinElmer) at 20× magnification. Subsequent image capturing was performed using the PerkinElmer Columbus Image Analysis System as previously described^{63 (link)}.