Ab33168

Cells were washed twice with DPBS (BI, China), and digested with Typsin (BI) for 1 min, folowed by wash with DPBS containing 0.5% BSA (0.5% PBSA). Next, cells were centrifuged and washed once with 0.5% PBSA. The resuspended cells in 200 μl 0.5% PBSA were passed the flow tube to obtain single cells. After that, cells were incubated with the antibodies for 30 min at room temperature. The antibodies used are Anti-NFATc1 (Abcam, ab2796); Anti-ISL1 (Abcam, ab178400); Anti-VE-cadherin (Abcam, ab33168), Anti-CD31 (R&D, FAB35679), Anti-KDR (R&D, FAB3579), Anti-CDH5 (BD Horizon, 561569), Anti-SOX9 (Proteintech, 67439-1-Ig, 1: 200), Anti-HEY1 (Proteintech, 19929-1-AP, 1: 200), Anti-JAG1 (Proteintech, 668909-1-Ig, 1: 200) or (Invitrogen, PA5-86057), Anti-NOTCH1 (Proteintech, 20687-1-AP, 1: 200), Anti-NOTCH4 (Abcam, ab225329, 1:100), Anti-P-SELECTIN (Proteintech, 60322-1-Ig, 1:200) and the isotype control antibodies: mouse IgG (R&D, C002P) and rabbit IgG (Invitrogen, 10500C). After three times washing, cells were treated with secondary antibodies at room temperature for 15 min. Finally, cells were washed twice and resuspended with 300–500 μl 0.5% PBSA, and then analyzed by Accuri C6 flow cytometer (BD Biosciences, USA).

Double immunofluorescence staining was performed as described previously64 65 (link). After blocking in 0.5% horse serum, sections were incubated with first antibodies anti-GFP (Rockland, 600-101-215,1:500, or Abcam, ab290,1:200), anti-Nestin (Aves Labs, NES, 1:100), anti-active RhoA-GTP (NewEast Bioscience, 26904, 1:100), anti-MMP3 (Abcam, ab52915, 1:100), anti-CD31 (Abcam, ab28364, 1:50), anti-αSMA (Abcam, ab5694, 1:200), anti-CD11b (Abcam, ab8878, 1:200), anti-VE-cadherin (Abcam, ab33168, 1:100), anti-Leptin Receptor (R&D, BAF497, 1:200), anti-fibronectin (Abcam, ab2413, 1:200), anti-collagen I (Abcam, ab21286, 1:200), anti-phospho-VEGFR2 (Tyr1175) (Abcam, ab38464, 1:100), followed by incubation with FITC or Cy3-conjugated secondary antibodies (Jackson ImmunoResearch). Nuclei were counterstained with 4,6-diamidino-2-phenylindole (Sigma). The sections were mounted with the ProLong Antifade Kit (Molecular Probes) and observed under a confocal microscope (FLUOVIEW FV300, Olympus).