Evans blue dye

Evans blue dye (Sigma, #E2129) was dissolved freshly in a sterile 0.9% NaCl/H₂O solution at a concentration of 20 mg/mL and sterile filtered. 100 µl of Evans blue solution was then injected into the lateral tail-vein of mice followed by an incubation of approximately 1 hr. Animals were then anesthetized, transcardially perfused with heparinized PBS solution and decapitated. The brain was carefully removed, weighted and snap-frozen in liquid nitrogen. The frozen tissue was then powderized under liquid-nitrogen protection in a standard mortar followed by careful resuspension in 1 mL 50% Trichloroacetic acid (TCA, Alfa Aesar, #A11156) in 0.9% NaCl. The mixture was then transferred to a gentleMACS C Tube (Miltenyi) and dissociated with the gentleMACS tissue dissociator (Miltenyi). Solid components were spun out, the supernatant was harvested and diluted at a ratio of 1:4 with 100% ethanol. Light emission at 680 nm after excitation at 620 nm was recorded with a plate reader (Tecan) and compared to a standard dilution series of Evans blue dye. Light emission was visualized with the Odyssey LTX imaging system (Li-cor).