Dextran-rhodamine 70-kDa (RHO70; 10mg/mL, 2.86µl/g/each; Sigma-aldrich, MO) and dextran-FITC 10-kDa (FITC10, 10mg/mL, 2.86µl/g/each; Sigma-aldrich, MO), or the fluorescently-labeled angiotensin II (AngIIfluo, 1µmol/L, 2.86µl/g/each; Anaspec, CA), were delivered intracarotidly, and allowed to circulate for 20 min. When indicated, hypertonic mannitol (1.4M, 2ml per 200–250g of animal; Sigma-aldrich, MO)22 (link)–24 (link) was infused intracarotidly 5 min prior to the dextrans. Hypothalamic and brainstem sections (40µm) were counterstained (Toto 1:50000).
Immunofluorescence (Table.S1) was performed for cell-type identification, and examined by confocal microscopy (LSM510, Zeiss Microimaging)25 (link). BBB permeability was assessed based on the detection of fluorescent-dye staining within brain microvasculature or parenchyma26 (link). The extravasated FITC10 signal was isolated using a digital subtraction procedure, and the proportion of FITC10-stained pixels in the subtracted image was calculated and compared among experimental groups. A densitometry analysis was used to compare differences in BBB protein constituents or immunoreactivity between WKYs and SHRs and results were expressed as % area within the nucleus occupied by the detected immunoreactivity25 (link).