Fluorescence Localization of Tight Junction Proteins and Stress Fibers in Endothelial Cells

Take a chambered slide containing a monolayer culture of rat brain microvascular endothelial cells, or RBMECs, mimicking the endothelial cell layer of the blood-brain barrier.

The cells exhibit disrupted tight junction proteins and actin stress fibers due to stress from oxygen-glucose deprivation followed by reoxygenation.

Remove the medium and wash the cells, apply a fixative to preserve cell architecture, then wash off excess fixative.

Treat with a detergent to permeabilize cellular membranes.

Incubate with a blocking agent to mask non-specific binding sites.

To label tight junction proteins, incubate with specific primary antibodies, then remove unbound antibodies.

Incubate with fluorophore-conjugated secondary antibodies to label the primary antibodies.

Alternatively, to label the stress fibers, add fluorophore-tagged phalloidin.

Wash to remove unbound labeling molecules.

Remove the chamber, apply a mounting medium containing a fluorescent DNA-binding dye to stain nuclei, then seal with a coverslip.

Using confocal microscopy, visualize the fluorescently labeled tight junction proteins and stress fibers.

Begin with exposing each well of RBMEC monolayers to 100 to 200 microliters of Opti