Isolation of Cerebral Capillary Amyloid Seeds

Brain capillaries were isolated from the cortices of 12-month–old rTg-DI rats, as previously described.^{27, 37} The isolated microvessels were analyzed microscopically by staining with thioflavin S and immunolabeling with an antibody to collagen IV to confirm the isolation of amyloid-containing microvessels. The remaining microvessels were treated with 3 mg/mL collagenase at 37°C overnight. The collagenase-treated samples were centrifuged at 16,000 × g for 5 minutes, and the resulting pellet was washed twice with tris-buffered saline. The amyloid pellet was resuspended in tris-buffered saline and again analyzed microscopically by staining with thioflavin S and immunolabeling with an antibody to collagen IV to confirm the isolation of vascular amyloid deposits and the absence of microvessels.