Mice bearing two-week old i.c. GL261 or K-Luc tumors were injected with TRITC-dextran 150 (Life Technology) solution (100 mg/kg i.v.). Brains were harvested after two hours, and fixed in paraformaldehyde for four hours before storage in 30% sucrose solution. Brains were then embedded in O.C.T. (Tissue-Tek), sectioned (10 μm) and baked at 37°C. Images were captured with an AX-70 fluorescent microscopy (Leica Microsystems Inc., Bannockburn, IL) and analyzed by Zeiss LSM Image Browser software.
For Evans blue permeability assay, tumor-bearing mice were anesthetized and injected with Evans blue dye (100 μL of a 1% solution in 0.9% NaCl; Sigma-Aldrich) into the retro-orbital plexus. Thirty minutes later, mice were sacrificed and perfused with PBS. Brains were removed, imaged and dried in 60°C overnight. The Evans blue dye was then extracted using 1 ml formamide at 55°C for 16 hours and quantified with a spectrophotometer at 630 nm (20 (link)).
For Evans blue permeability assay, tumor-bearing mice were anesthetized and injected with Evans blue dye (100 μL of a 1% solution in 0.9% NaCl; Sigma-Aldrich) into the retro-orbital plexus. Thirty minutes later, mice were sacrificed and perfused with PBS. Brains were removed, imaged and dried in 60°C overnight. The Evans blue dye was then extracted using 1 ml formamide at 55°C for 16 hours and quantified with a spectrophotometer at 630 nm (20 (link)).