Tissue collection, processing and immunostaining

Upon development of neurological symptoms, mice were deeply anesthetized before perfusion with cold DPBS followed by 1% PFA. Brains were rapidly dissected in cold DPBS and then fixed in 100% MeOH at 4 °C overnight. Samples were rehydrated at 4 °C in DPBS for 4 h before embedding in 3% low-melt agarose gel (IBI scientific, #{"type":"entrez-nucleotide","attrs":{"text":"B70051","term_id":"2709275","term_text":"B70051"}}B70051). 150 um thick free-floating sections were cut using a Leica vibratome. Floating sections were incubated in blocking solution (PBS + 0.5% Triton x-100 + 10% normal donkey serum) at RT for 30 min before incubating with primary antibodies at 4 °C overnight. The next day sections were washed in DPBS, transferred to blocking solution containing the appropriate secondary antibodies (1:500) and incubated at room temperature for 2 h. Finally, sections were incubated in Hoechst (1:1000 in PBS) for 10 min before final DPBS washing and mounting onto slides (Fisher, Superfrost), and coverslipped (Prolong Gold Antifade, ThermoFisher). Primary antibodies used in this study include: eGFP (Aves, #GFP1020), CD31 (BD Biosciences, #550,274), Glut1 (Millipore, #07–1401), Collagen IV (BioRad, #161,115), Desmin (Cell Signaling, #5332), human Vimentin (eBioscience, #11–9897-82), Claudin-5 (Thermofisher, #352,588), Plvap (BD Biosciences, #550,274), Ter119 (Invitrogen #14–5921-82) and Hoechst (ThermoFisher). Corresponding secondary antibodies used were all purchased from Jackson ImmunoResearch. Images were acquired on a confocal microscope (Nikon A1), and image analysis was performed in NIH Image J. Statistical analyses of these data were performed in GraphPad prism as described in the manuscript. P-values of ≤ 0.05 were considered statistically significant.

For histology, mouse brain tissue was fixed in 10% formalin overnight and transferred to 70% ethanol before paraffin embedding. 5 μm thick sections were prepared on a microtome (Lecia), and processed for hematoxylin–eosin (H&E) staining. For immunohistochemistry of primary patient samples 5 µm thick paraffin sections were prepared and stained using standardized techniques. Primary antibodies used include anti-CD31 (Dako, #M0823), anti-Cldn5 (Invitrogen, #34–1600) and anti-Glut1 (Millipore, #07–1401). Stains were developed with secondary HRP and DAB immunoreactivity secondary kits (Dako) and counterstained with Hematoxylin before mounting.