Goat anti mouse alexa fluor 488 af488

The pHAECs or iHAECs grown in 1% gelatin-coated tissue culture plates up to about 90% confluence were collected with TE/TN. Cells were washed, fixed, permeabilized using the Fix/Perm kit (Invitrogen Corporation, Frederick, MD), and incubated with FcBlock (BD Biosciences, San Jose, CA) and 10% goat serum to block nonspecific staining. Cells were stained with the primary antibodies for 2 h on ice, washed, and incubated with the secondary fluorescently labeled antibodies for 2 h on ice, followed by flow cytometry analysis (BD LSR II; Becton Dickinson, Franklin Lakes, NJ). Data were analyzed with FlowJo (FlowJo, LLC, Ashland, OR). The following antibodies were used: mouse anti-human CD31 (clone JC/70A; Pierce/Thermo Fisher Scientific, Rockford, IL); CD40 (clone 5C3; BD Biosciences); MHC-II (clone TÜ39; BD Biosciences); VE-cadherin (clone 123413; R&D Systems, Minneapolis, MN); rabbit anti-human ICAM-1 (polyclonal antibody; NeoScientific, Woburn, MA); CD14, eNOS, TLR4, vWF, and ZO-1 (all polyclonal antibodies; Bioss Antibodies, Inc., Woburn, MA); and secondary antibodies goat anti-mouse Alexa Fluor 488 (AF488) and goat anti-rabbit Alexa Fluor 647 (AF647) (both from Molecular Probes, Eugene, OR).