K5001

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. 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).

Tumor samples were routinely obtained by open surgical biopsy. Formalin-fixed paraffin-embedded (FFPE) tumor sections were stained with the HLA-G specific mAb clone 4H84 (Exbio, 11-499-C100), which detects all HLA-G isoforms using Autostainer (Dako, 3400-9580-03). Biotinylated secondary antibodies were visualized with red chromogen or brown 3,3’-diaminobenzidine tetrahydrochloride (DAB) for HLA-G or CD3 according to the manufacturer (Dako K5001, K5005). CD3 quantification (Thermo Scientific, RM-9107-S1) was done on serial tissue sections corresponding to HLA-G^pos/neg tumor biopsies, along with expert histomorphology review by an experienced pathologist. Cytomorphological features, particularl nuclear size and shape as well as chromatin quality were thoroughly assessed to differentiated between (larger and atypical) EwS cells and infiltrating lymphocytes. Biopsies of insufficient size to count CD3 cells in 4-5 high power fields (HPF; magnification 400x) were excluded. Corresponding areas of noticeable HLA-G expression on tumor cells where quantified only if HLA-G expression was clearly separable between EwS and infiltrating lymphocytes. HLA-G^pos areas with strong infiltration, bleeding and necrosis as well as intratumoral stroma where excluded. A 200x magnification was used for all images.