Axioplan 2 fluorescent microscope

Mouse brain endothelial cells were seeded on 1% fibronectin–coated Nunc Lab-Tek II Chamber Slides (Thermo Scientific) in DMEM. After siRNA treatment or treatment with Aβ(1–40) monomer or Aβ(1–40) dimer peptides, cells were fixed with 4% paraformaldehyde (PFA; pH 7.4); blocked with 5% normal goat serum (NGS); and incubated with polyclonal rabbit anti–claudin-5 (1:100), polyclonal rabbit anti-occludin (1:100), polyclonal rabbit anti–ZO-1 (1:100), or phalloidin–Alexa Fluor 488 (1:300; all from Invitrogen) overnight at 4°C. Cells were then incubated with Cy3-conjugated goat anti-rabbit secondary antibody (1:500; Abcam) for 3 hours at room temperature and counterstained with 4′,6-diamidino-2-phenylindole (DAPI; 1:5000). Mouse brain cryosections (12 μm thick) were permeabilized with 0.5% Triton X-100; blocked with 5% NGS; and incubated overnight with tight junction primary antibody, polyclonal rabbit anti–amyloid-β AW7 antibody (1:1000), or isolectin IB4–Alexa Fluor 488 (1:300; Invitrogen), as described (18 (link), 19 (link)). Sections were then incubated with Cy2- or Cy3-conjugated goat anti-rabbit IgG secondary antibody (1:500; Abcam) for 3 hours at room temperature and counterstained with DAPI. Analysis of stained cells or brain cryosections was performed using a Zeiss Axioplan 2 fluorescent microscope or an Olympus FluoView FV1000 confocal microscope with integrated software.