Ve cadherin

Anti-CD31 antibody was from Transduction Laboratories (Lexington, KY). Anti-AQP5, –SPB, -β-catenin antibodies were from Abcam (Cambridge, MA). Anti-β-actin monoclonal antibody was from Sigma (St. Louis, MO). Anti-VEGFR2 and LRP5 antibodies were from Cell Signaling (Danvers, MA). Anti-Tie2 monoclonal antibody was from Upstate (Lake Placid, NY). Anti-Tie2 polyclonal antibody was from Santa Cruze Biotechnology (Dallas, TX). Anti-EpCAM, -CD31, -VE-cadherin, and –CD45 antibodies were from BioLegend (San Diego, CA).

The peripheral blood mononuclear cells were suspended in saline and incubated with fluorescein isothiocyanate anti-mouse stem cells antigen (Sca)-1 (Invitrogen, 11-5981-82, Carlsbad, CA, USA) and phycoerythrin anti-mouse vascular endothelial growth factor receptor 2, also known as Flk-1 (Invitrogen, 12-5821-82, Carlsbad, CA, USA) at room temperature for 30 min. A BD FACScalibur flow cytometer (BD, East Rutherford, NJ) was used, and data were analyzed with FloJo (Treestar). Data are presented as % gated, relative to control group.
EPCs were harvested and washed prior to suspension in phosphate-buffered saline (PBS) for flow cytometry. To identify the characteristics of EPCs, VE-cadherin (Biolegend, 348505, San Diego, CA), CD31 (Biolegend, 303117, San Diego, CA), CD34 (BD, 555821, East Rutherford, NJ), KDR (R&D, FAP357, Minneapolis, MN, USA), CD133 (MACS, 130-111-756, Germany), CD3 (Biolegend, 300407, San Diego, CA), CD68 (Biolegend, 333805, San Diego, CA), CD86 (Biolegend, 374203, San Diego, CA), CD163 (Biolegend, 33605, San Diego, CA), and CD206 (Biolegend, 321123, San Diego, CA) antibodies were used. Cells were analyzed by BD FACScalibur flow cytometer (BD, East Rutherford, NJ), and data were analyzed with FloJo (Treestar). Data are presented as % gated.

For immunofluorescence on cultured cells, cells were fixed with 4% PFA for 15 min, permeabilized in PBS-Triton 0.1% (Sigma) for 10 min and incubated in 5% normal goat serum for 1 hr. The following primary antibodies were used: ZO-1 (Rabbit, 61–7300; Thermo Fisher Scientific), VE-Cadherin (mouse, 348502; BioLegend), CD63 (rat, D623-3; MBL), RalA (mouse, 610221; BD), RalB (mouse, 04037; Millipore), CD146 (Mouse, P1H12, Thermofisher). The following secondary antibodies were used: goat anti-mouse/rat/rabbit coupled with Alexa Fluor 488, Alexa 555, or Alexa 647 (Invitrogen). Cells were mounted with DAPI-containing Vectashield (Vector Laboratories).
For immunofluorescence on tissue sections, tissues were cut in 7-μm-thick sections, dewaxed for paraffin-embedded tissues and air-dried for frozen tissues. Sections were incubated first in 5% normal goat serum for 2 hr in a humidified container. The following antibodies were used: CD31 (Mouse, 37–0700; Thermo Fisher Scientific), S100A4 A gift from Nona Ambartsumian (Institut for Cancer Biology, Copenhagen, DK-2100, Denmark.), F4/80 (Rat, ab6640; abcam), rabbit monoclonal antibody against Ki67 (Rabbit, RM-9106-S0; Thermo Fisher Scientific), and caspase-3 (Mouse, 966S1; Cell Signaling Technology). Secondary antibodies were similar to the ones used with cells. Nuclei were stained with DAPI (Sigma).