Mice were sacrificed under deep anesthesia by intracardial perfusion with PBS followed by perfusion with 4% w/v paraformaldehyde solved in PBS. Brains, spinal cords, optic nerves, kidneys and spleens were removed and fixed in 4% paraformaldehyde overnight. Cervical, thoracic, and lumbar spinal cord was cut into 11–12 4 mm thick transverse segments prior to embedding; 5 μm thick sections were stained for hematoxylin and eosin (H&E) and Luxol-fast blue/periodic acid-Schiff. Immunohistochemistry was performed using a biotin-streptavidin peroxidase technique (K5001; Dako) and an automated immunostainer (AutostainerLink 48; Dako). Sections were pretreated in a steamer (treatment solutions pH 6.0 or 9.0; Dako) before incubation with the primary antibodies anti-Mac3 (clone M3/84, 553322, 1:100; BD Pharmingen), anti-CD3 (MCA 1477, 1:50; Serotec), and anti-AQP4 (HPA014784, 1: 4000; Sigma). DAB was used as a chromogen and sections were counterstained using hematoxylin.
To quantify the perivascular loss of immunoreactivity against AQP4, three consecutive brain sections per mouse were analyzed using Image J (NIH) [49 (link)]. The sizes of areas showing AQP4 loss and the corresponding vessel lumen were measured and processed as individual ratios (any ratio > 1 indicates perivascular AQP4 loss).
To quantify the perivascular loss of immunoreactivity against AQP4, three consecutive brain sections per mouse were analyzed using Image J (NIH) [49 (link)]. The sizes of areas showing AQP4 loss and the corresponding vessel lumen were measured and processed as individual ratios (any ratio > 1 indicates perivascular AQP4 loss).