Bz 2 analysis application

HAECs grown on glass coverslips were washed with PBS and fixed with 10% (v/v) formalin for 15 min. For detection of E-selectin and RelA/p65, we permeabilized fixed cells with 0.2% (v/v) Triton X-100 and blocked with PBS containing 10% (v/v) normal goat serum. The cells were then incubated with an anti-E-selectin antibody (1:200) and anti-RelA/p65 antibody (1:200) at 4 °C overnight. After washing, cells were incubated with Alexa Fluor 532-conjugated secondary (1:100; Molecular Probes, Eugene, OR, USA) for 1 h. Nuclei were visualized using Hoechst dye. Images were observed on a BZ-9000 fluorescence microscope (Keyence, Osaka, Japan) using BZ-II; analysis application (Keyence, Osaka, Japan).

Cells or tissues were fixed with 4% paraformaldehyde. Tissues were frozen and cryosections were used. Primary antibodies shown in Table 1 were applied on cells or cryosections followed by appropriate secondary antibodies conjugated with Alexa Fluoro 488 and/or 594 (Molecular Probes), also listed in Table 1, and mounted with Permafluor™ Aqueous mounting medium (TA-030-FM; Thermo; Fremont, CA) just after being counter-stained with DAPI (71-03-01 KPL; Gaithersburg, MD). Samples were imaged using a microscope BZ-9000 (KEYENCE; Osaka, Japan; www.keyence.co.jp), and were recorded and analyzed with a BZ-II analysis application (KEYENCE).