*2.1.7 Measuring intracellular calcium and sodium concentrations*

Intracellular calcium was quantified in cultured cerebral capillary endothelial cells by using a fluorescent probe and confocal laser microscopy, as previously described by us [39]. Cells were preloaded with 5 μM Fluo-4 [39] and treated under conditions of ischemia and reperfusion as described above. Calcium concentration was quantified by measuring emitted fluorescence [39, 40] at a wavelength of 494 nm in 50 randomly chosen (computer-assisted) cells, representing each treatment.

Intracellular sodium in cultured cerebral capillary endothelial cells was measured as previously described by us [39]. Cells were pre-treated with Sodium Green (5 μM), and the fluorescent signal was quantified by fluorescence microscopy. Measurements were made from 50 randomly chosen cells representing each treatment.

### *2.1.8 Measuring actin stress fibers in cultured cells*

Cerebral capillary endothelial cells were grown on coverslips and exposed to conditions simulating normoxia, ischemia, and reperfusion as described above. Following treatment, the monolayers were washed in phosphate buffered saline (pH 7.4), fixed for 5 minutes in 3.7% buffered formaldehyde at room temperature, and rinsed again with the buffer [41]. A mixture of phalloidin (0.05 mg/ml buffer) and 1% dimethyl sulfoxide was added to the cells for 40 minutes at room temperature, in a humidified chamber. Following staining, the coverslips were washed with buffer and mounted in a mixture of 30% glycerol in 70% buffer (vol/vol). To determine the effects of calcium-mediated cytoskeletal activation, the cells were incubated in the presence of a myosin light chain kinase inhibitor (0.1 μM, Sigma).

The tissue was observed and evaluated using laser confocal microscopy, at a thickness of 4 microns.
