Af2335

Brains were sectioned sagittally using a vibratome at either 50 μm (one-hour post-fixed tissue) or 30 μm (tissue post-fixed overnight). At least three non-adjacent sections (> 100 μm apart) were analysed per animal per condition. Sections were processed as floating sections. Sections which had been post-fixed overnight underwent antigen retrieval (incubated in 0.25% trypsin for 5 min at 37 °C, followed by 3 × 5 min washes in PBS). Sections were blocked for 2 h at room temperature on an orbital shaker in blocking solution (5% donkey serum, 0.1% triton in PBS). Sections were incubated with primary antibodies [Fibrinogen (1:5000; Dako; A0080), Glut-1 (1:500; ThermoFisher; MA5–11315), Glut-1 (1:10,000; Millipore; 07–1401), PDGFR-β (1:500; eBioscience; 14–1402-82), Aminopeptidase-N/CD13 (1:250; R&D Systems; AF2335)] in blocking solution overnight at 4 °C on a shaker. Sections were then washed in PBS (3 × 5 min) before being incubated with Alexa Fluor secondary antibodies for 4 h at room temperature on a shaker. Sections were washed in PBS (3 × 5 min), incubated with DAPI (1 min at room temperature), further washed in PBS (3 × 5 min), then mounted on slides with Dako Mounting Medium.

Mice were anesthetized with pentobarbital sodium and transcardially perfused with PBS containing 5 U/ml heparin. Brains were dissected, cryosectioned with a thickness of 14–18 mm, and fixed in ice-cold acetone. Sections were blocked with 5% normal goat serum for 1 h at room temperature and incubated overnight with primary antibody goat anti-CD13 (R&D Systems; AF2335; 1:200) which was diluted in blocking solution at 4 °C. Sections were washed in PBS and incubated with the secondary antibody Alexa 568-conjugated donkey anti-goat (Invitrogen; A11057; 1:200). Further, sections were stained with Dylight 488-conjugated tomato lectin (DL-1174, 1:100, Vector Laboratories) and coverslipped with fluorescent mounting medium (Dako, Carpinteria, CA, USA) to visualize brain microvessel with a 510 confocal microscopy (Zeiss). For thioflavin-S staining, brain sections were stained for 10 min with 0.2% thioflavin-S (T1892, Sigma-Aldrich) diluted in PBS. After repeated wash with PBS, brain sections were imaged using an IX53 fluorescence microscope (Olympus). The quantification of images was analyzed using the Image J software.