Sp5 aobs confocal laser microscope

Permeability visualization experiments were performed on 18×18 mm square Rinzle plastic coverslips with In vitro Vascular Permeability Imaging Assay (17-10398, Merck, UK). HUVEC cells were cultured for 48-72 hours on biotinylated gelatin coated coverslips in 35 mm culture dishes in a density of 4.5× 10 5 cells/ dish. The culture medium was then changed to EGM-2 containing 2% FBS for 2 hours prior to stimulation. Then culture medium was mixed with test samples at 1:3 ratio and incubated for 30 min at 37°C before evaluation of HUVEC cell monolayer permeability. FITC-streptavidin (25 μg/ml) was directly added to the culture medium for 5 min, followed by two washing steps (3 ml of PBS, pH 7.4, 37°C). The cells were fixed (3.7% formaldehyde in PBS for 10 min) and subjected to immunofluorescence staining for VE-cadherin according to the manufacturer's instructions. Overlay of FITC and VE-cadherin staining demonstrates reciprocal relations between VE-cadherin peripheral localization and increased local permeability of FITCstreptavidin. Local permeability change induced by conditioned medium was visualized in cells grown on biotinylated gelatin plastic coverslips. The pattern of FITC-streptavidin binding to the biotinylated gelatin underlying the cell monolayer was examined under Leica SP5-AOBS confocal laser microscope. Images were processed with ImageJ software.