Clone 11d4

Single-cell suspensions from the AGM region were prepared by dispase/collagenase-mediated dissociation. Antibodies used for staining of cells were anti-CD45-BV450 (BD Horizon, clone 30F11, 1:100), anti-CD45-AF700 (1:100, clone 30F11, BD pharmingen), anti-VE-cadherin-AF647 or -AF488 (1:100, Clone eBioBV13, Biolegend) and biotinylated anti-VE-Cadherin (1:50, clone 11.D4.1, Pharmingen), followed by incubation with streptavidin-PE (1:600, BD Pharmingen), anti-CD43-PE (1:200, clone eBioR2/60, eBioscience), anti-cKit-APC (1:100, clone 2B8, eBioscience), anti-CD31-PE (1:200, MEC13.3, Pharmingen), anti-Sca1-V500 (1:100, clone D7, BD Bioscience), anti-CD41-BV421 (1:100, clone MWreg30, Biolegend) and anti-Ter119-PerCp-Cy5.5 (1:100, clone TER119, eBioscience). 7-Aminoactinomycin D viability staining solution was used to exclude dead cells, and gates were set using appropriate fluorescence minus one controls. Sorting was performed on a FACSAriaII using the FACSDiva software.

Plasma from C57BL/6J mice was diluted 1:25 in PBS and analyzed by Western-blotting. Proteins were separated by 8% SDS-PAGE and transferred onto a nitrocellulose membrane using a Trans–Blot turbo transfer system. Membranes were incubated with rat anti–mouse primary antibody to VE-cadherin (0.25 μg/mL, Clone 11D4.1, reference 550548, BD Biosciences, Franklin Lakes, NJ, USA). After a rabbit anti-rat secondary antibody (1:2000) incubation (reference ab6734, Abcam, Cambridge, United Kingdom), membranes were revealed by chemiluminescence using a ChemiDoc XRS+ imager system. Nitrocellulose membranes, the transfer system, enhanced chemiluminescence detection reagents and the detection system were from Bio-Rad Laboratories (Marnes-la-Coquette, France). Bands were quantified by densitometry by using the public domain ImageJ software ). We also quantified sVE in 12 plasma samples from a pilot experiment from 6 N-exposed and 6 IH-exposed mice treated with the vehicle only (vehicle is PBS).