D3312

We used dextran to measure BBB permeability as described in previous studies with modifications (1 (link), 22 (link)). Briefly, at 3, 6, and 12 h after burns, each mouse was injected with 100 µl 10-kDa dextran tetramethylrhodamine lysine fixable (4 mg/ml, catalog number: D3312, Invitrogen, Carlsbad, CA, USA) and 70-kDa fluoro-Ruby dextran tracer (555/580) (4 mg/ml, catalog number: D1818, Invitrogen, Carlsbad, CA, USA), a high molecular weight tracer, through the tail vein. After 10 min, each mouse was decapitated, brain tissue was harvested, and fixed using 4% paraformaldehyde (PFA) overnight at 4°C. The brain tissues were cryopreserved in 30% sucrose for 1 day and frozen in Tissue-Tek OCT (Sakura). BBB permeability was detected by immunohistochemistry as described previously with modifications (1 (link), 22 (link)). The frozen mouse brain hemispheres were cut into 12 µm sections and used for immunohistochemistry staining. These sections were postfixed in 4% PFA at 20–25°C for 15 min, washed in phosphate buffer saline (PBS) three times for 10 min, and permeabilized with 1% Triton X-100 for 10 min. Then, these sections were blocked with 10% albumin from lowlenthal serum for 1 h and incubated with isolectin B4 (1:200; catalog number: I21411, Molecular Probes, San Francisco, CA, USA) overnight at 4°C for immunohistochemistry imaging of blood vessels.