Mouse BMEC cells were seeded in 4-chamber polystyrene vessel tissue culture treated glass slides (Corning, NY) and infected with Ad. LacZ or Ad. KLF11. Seventy-two hours after infection, BMEC cultures were subjected to OGD treatment and reperfusion for indicated time points. The cells were then fixed in 4% paraformaldehyde followed by blocking with 5% normal goat serum in PBST for 1 h at room temperature. The cells were then incubated with the following primary antibodies overnight at 4 °C: mouse anti-ZO-1 (1:100; Invitrogen, Carlsbad, CA), rabbit anti-Occludin (1:100; Thermo Fisher Scientific, Waltham, MA). After rinse in PBS, cells were incubated with secondary antibodies: Alexa Fluor 488-conjugated goat anti-rabbit IgG, and goat anti-mouse IgG (all at 1:400; The Jackson ImmunoResearch Laboratories). After PBS rinses, cells were counterstained with DAPI for nuclear labeling and mounted with antifade Vecta-Shield solution (Vector Laboratories). Images were collected on an Olympus FV1000-II confocal microscope and processed in Adobe Photoshop for compositions (17 (link)). Immunofluorescence intensities of ZO-1 and occludin were quantified by ImageJ. Three ROIs were randomly selected from each chamber, and 2-3 chambers were quantified for each experimental condition in each independent culture.