Ab236284

IH tissues were obtained from fresh surgical specimens of three patients, the age of the three patients was less than 6 months. The diagnosis of IH of these patients was made based upon clinical examination and computed tomography with and without contrast in the West China Hospital, West China Medical School, Sichuan University China. The study was approved by the West China Hospital, West China Medical School, Sichuan University China Ethics Committee and the consents were signed by the parents of those three patients. The tumor tissues were cut into 2 × 2 mm pieces and placed in a digestive solution containing 0.2% collagenase A for digestion. Cells with positive expressions were used as the specific markers of factor VIII (anti-factor VIII, rabbit; 1:100; ab236284; Abcam), CD31 (anti-CD31, rabbit; 1:50; ab28364; Abcam) and CD34 (anti-CD34, rabbit; 1:100; ab81289; Abcam) hemangiomas were sorted by using flow cytometry. The positive cells were incubated with EGM-2V (Lonza, U.S.A.) containing 20% FBS (Gibco, Solarbio, Beijing), SingleQuot (Hefg, VEFG, hFGF-B, ascorbic acid, heparin and gentamicin/amphotericin) and PS at 37°C in 5% CO₂.

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)}.