Assessment of blood-brain barrier permeability
Blood-brain barrier permeability was assessed by staining brain sections for endogenous immunoglobulin type G (IgG). Whole brains were snap-frozen in liquid nitrogen and stored at -80 °C. Ten µm coronal sections were cut and thaw-mounted onto Superfrost Ultra Plus slides (Thermo Fisher Scientific, USA). Frozen brain sections were air-dried (5 min), fixed with 100 % acetone at 4 ˚C (10 min) and washed 3 x 5 min with 0.01 M PBS. Sections were incubated with goat anti-mouse IgG (goat anti-mouse IgG (ab150118), Alexa 555, 1:200 dilution) (Abcam, Cambridge, UK) in antibody diluent (3 h), then washed 3 x 5 min with 0.01 M PBS in a dark room. Sections were mounted with VECTASHIELD®mounting medium containing the nuclear stain DAPI (Vector Laboratories, Inc. Burlingame, USA) and cover slipped. Edges were sealed with nail polish and sections were stored at 4 ˚C until imaging. The hippocampus and cortex (2 images per animal) were imaged with an Olympus DP73 Camera (Olympus Corporation, Tokyo, Japan) connected to an Olympus BX53 microscope (Olympus Corporation, Tokyo, Japan) at 100x and 200x magnification running CellSens Standard Software (version 1.17, Olympus Corporation). Exposure settings, ISO and black balance were kept consistent across all images. Percentage area of staining was analysed using FIJI (National Institute of Health, USA) and the threshold settings were kept consistent across all images.