This article describes a method for visualizing tight junction proteins and actin stress fibers in rat brain microvascular endothelial cells (RBMECs) under conditions of oxygen-glucose deprivation. The protocol includes cell fixation, permeabilization, and immunofluorescence labeling, followed by confocal microscopy imaging.
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-MEM reduced serum media, and continue their incubation for an hour. Then wash the chamber slides three times, using 100 to 200 microliters of PBS per well per wash. Fix the cells with 4% paraformaldehyde in PBS, and incubate the slides for 15 minutes at room temperature. And wash the cells off three times with PBS as before.
Now, permeabilize the cells by applying 100 to 200 microliters of 0.5% Triton X-100 in PBS to each well. And let the plate incubate for another 15 minutes at room temperature. After the cells have been permeabilized, apply the blocking solution. And let this act on the cells for an hour at room temperature.
Next, either perform immunofluorescence for ZO-1 protein or label F-actin. To conduct immunofluorescence of the cells for ZO-1, incubate them with the primary antibody overnight at 4 degrees Celsius. The next day, wash the cells three times in PBS, such as when removing the fixative. Then incubate the cells in 100 to 200 microliters of FITC tag anti-rabbit secondary antibody for an hour at room temperature, to label the cells with rhodamine phalloidin.
After applying the blocking solution for an hour, expose the cells to 100 to 200 microliters of rhodamine phalloidin in a 1 to 50 dilution, with 2% BSA PBS for 20 minutes. After the secondary antibody or rhodamine phalloidin incubation, wash the cells with PBS three times again as before. Then remove the chambers by peeling off the elastic. Proceed with mounting the slides, using medium containing antifade and DAPI. Image the cells with confocal microscopy in a single plane with a 60x objective.
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