Ma5 13021

SMCs, SMC-ECs, and HUVECs were seeded and grown in 4-well chamber slides. They were stimulated with 10ng/ml TNFα for 6 hours before immunofluorescence stained with ICAM-1 antibody (MA5-13021, ThermoFisher Scientific).

Cells were seeded on glass slide and cultured in corresponding media until achieved 80% confluency. After washing with PBS, cells or frozen sections were fixed with 4% paraformaldehyde (PFA) (Sigma-Aldrich) for 15 minutes and then permeabilized with 0.1% Triton X-100 (Sigma-Aldrich) for another 15 minutes. Samples were blocked with 5% appropriate serum/1 × PBS at 37 °C for 30 minutes followed by incubation with primary antibodies at 37 °C for 1 hour: CD34 (sc7324, Santa Cruz, 1:50); CD31 (sc1506, Santa Cruz, 1:50); eNOS (610297, BD Biosciences, 1:50); CD144 (sc6458, Santa Cruz, 1:50); KDR (ab9530, Abcam, 1:100); human specific CD31 (ab32457, Abcam, 1:100); HES5 (Ab5708, Millipore, 1:50); vWF (ab6994, Abcam, 1:100); VCAM-1 (sc-13160, Santa Cruz, 1:50); ICAM-1 (MA5-13021, ThermoFisher, 1:50). After washing with PBS, samples were incubated with the appropriate Alexa Fluor-conjugated secondary antibodies (Invitrogen, 1:500) for 45 minutes at 37 °C. Samples were washed with PBS and then DAPI (Sigma Aldrich) was applied for 3 minutes. The slides were mounted with Fluorescent Mounting Media (Dako) and images were acquired by Olympus IX81 microscope equipped with TRITC, FITC and DAPI filters.