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.