Alexa 555 conjugated goat anti rabbit antibody

To visually determine whether nanoparticles damage the tight junctions of the intestinal tissue cultures, we immunostained the epithelial co-culture for the tight junction protein occludin and imaged the cell layers with a confocal fluorescence microscope. After 24 hours of exposure to nanoparticles, the cells in the transwells were washed with PBS three times and fixed in situ with 2% paraformaldehyde, rinsed with PBS containing 1% bovine serum albumin, permeabilized with 0.1% Triton X-100, and then immunostained with an antibody against occludin (rabbit anti-human occludin, 2 μg/mL, Invitrogen Inc., Eugene, OR) for 40 minutes at room temperature (at 0.04 μg/mL). After washing, fluorescent secondary antibodies (Alexa-555-conjugated goat anti-rabbit antibody, 250μg/mL, Invitrogen Inc., Eugene, OR) were added at a concentration of 1.25 μg/mL for 40 minutes in the dark at room temperature. Cultures incubated with the rabbit IgG (0.04 μg/mL) and secondary goat anti-rabbit IgG (Invitrogen Inc., Eugene, OR) served as negative immunofluorescent control (at a concentration of 1.25 μg/mL). Images were captured using a Leica SP2 confocal microscope (Leica Microsystems, Bannockburn, IL).
Transmission electron microscopy (TEM): Samples were coated with carbon on a TEM grid and imaged with a FEI T12 spirit TEM system at the Cornell Center for Materials Research.