Evans blue staining (EB)

The mice were transcardially perfused (via the left ventricle) with PBS 6 h post intravenous injection of 2% EB (Sigma-Aldrich) following 24 h post-injection of MPC-n(BSA). The brains were harvested, photographed using a digital camera, fixed in 4% PFA, and sectioned to ~5-8-μm-thick slices. The permeability of EB was observed using the Olympus FluoView 1200 confocal microscope (Olympus) at 680 nm upon excitation at 620 nm. To quantify the amount of EB staining, we collected the ipsilateral hemispheres from each group that were weighed, homogenized in 500 µL of N-dimethylformamide (Sigma-Aldrich), and centrifuged at 10,000 rpm for 30 min at 4 ℃. Next, the supernatants were collected, and absorbance was measured at 620 nm using a microplate reader.