Alexa fluor 546

A total of 3×10³ transfected Kasumi-3 and Kasumi-6 cells were transferred to each well of a 96-well plate in 0.1 ml medium, and cultured at 37°C for 48 h before the addition of BrdU. The experiment was conducted using the BrdU Cell Proliferation Assay (cat. no. QIA58; Sigma-Aldrich; Merck KGaA) according to the manufacturer's protocol. Then, cells were cultured with 20 µl/well diluted BrdU reagent (10 mM) for 6 h, fixed with Fixing solution and incubated for 30 min. After fixation, the cells were incubated for another 48 h with peroxidase-coupled anti-BrdU-antibody (supplied by the kit), followed by washing twice with ice-cold PBS. After incubation with peroxidase substrate for 3 h, OD values were measured at 450 nm. For the represent images, the anti-Brdu (1:500; cat. no. ab6362; Abcam) was used as primary antibody, and Goat anti-rabbit Alexa Fluor^® 546 (1:2,000; cat. no. A11010; Invitrogen; Therno Fisher4 Scientific, Inc.) was used as the secondary antibody. The detailed method has been previously published (21 (link)).

HUtMEC were cultured on glass slides, fixed with cold acetone, and blocked by 5% FBS in PBST. They were incubated with anti-Ang-2 (rabbit polyclonal; proteintech TM; cat. no. 24613-1-AP) and anti-VE-cadherin (rabbit polyclonal, Cell Signaling; cat. no. 2500) at 4°C. Cells were incubated with anti-rabbit IgG conjugated with Alexa Fluor^®546 (Abcam; cat. no. ab60317) and anti-human Ig G conjugated with Alexa Fluor^®488 (Abcam; cat. no. ab150129). Nuclei were stained with 4′,6-diamidino-2-phenylindole phenylindole (Enzo; cat. no. BML-AP402). Afterward, the samples were mounted in fluorescent mounting medium (DAKO; cat. no. S3023) and immunofluorescent images were acquired using a Zeiss LSM510 confocal fluorescence microscope (Carl Zeiss).