Sorted Flk1+ cells were plated on gelatin-coated dishes in the differentiation medium. For induction to an endothelial cell, differentiation medium supplemented with 100 ng/mL human VEGF165 (R&D Systems, Minneapolis, MN) was used. The medium was replaced every 2 days. After a 4-day culture, cells were fixed with 4% PFA (Wako Pure Chemical, Osaka, Japan) for 20 minutes at 4°C.
Then, specimens were blocked with 3% goat serum (Vector Laboratories, Burlingame, CA) with PBS (GIBCO) for 30 minutes at room temperature, and the following primary antibodies were applied to the sections at 4°C overnight: rabbit polyclonal anti-α-SMA antibody (1 : 200; Santa Cruz Biotechnology Inc., Santa Cruz, CA) or rabbit polyclonal anti-PECAM antibody (1 : 200; Santa Cruz Biotechnology Inc.). After washing, the following secondary antibodies were loaded for 1 hour at room temperature in a dark box: Alexa Fluor 488-coupled goat anti-rabbit IgG antibody (1 : 200; Invitrogen) or Alexa Fluor 594-coupled goat anti-rabbit antibody (1 : 300; Invitrogen). Finally, nucleus staining was performed using DAPI (Merck, Tokyo, Japan). The stained sections were observed using a fluorescence microscope (BX51, Olympus Optical, Tokyo, Japan) and images were obtained by a CCD camera (DP70, Olympus Optical).
Then, specimens were blocked with 3% goat serum (Vector Laboratories, Burlingame, CA) with PBS (GIBCO) for 30 minutes at room temperature, and the following primary antibodies were applied to the sections at 4°C overnight: rabbit polyclonal anti-α-SMA antibody (1 : 200; Santa Cruz Biotechnology Inc., Santa Cruz, CA) or rabbit polyclonal anti-PECAM antibody (1 : 200; Santa Cruz Biotechnology Inc.). After washing, the following secondary antibodies were loaded for 1 hour at room temperature in a dark box: Alexa Fluor 488-coupled goat anti-rabbit IgG antibody (1 : 200; Invitrogen) or Alexa Fluor 594-coupled goat anti-rabbit antibody (1 : 300; Invitrogen). Finally, nucleus staining was performed using DAPI (Merck, Tokyo, Japan). The stained sections were observed using a fluorescence microscope (BX51, Olympus Optical, Tokyo, Japan) and images were obtained by a CCD camera (DP70, Olympus Optical).