Evaluating Cerebral Vasculature Permeability

To evaluate the permeability of the cerebral vasculature, we injected 2% Evans Blue (Sigma-Aldrich, Hamburg, Germany) tracer diluted in 0.9% NaCl (Fresenius Kabi, Bad Homburg, Germany; i.v.) 1 h after the induction of tMCAO. After 24 h, the animals were euthanized and brain tissue was removed. The tissue was cut in coronal sections (2 mm thick) that were incubated in 4% paraformaldehyde (Sigma-Aldrich, Hamburg, Germany) in the dark for 30 min and then divided into ipsi- and contralateral areas. Brain sections were weighed and incubated with formamide (Sigma-Aldrich, Hamburg, Germany) at 50 °C in the dark for the following 24 h. On the next day, samples were centrifuged for 20 min and 50 µL of the supernatant was collected and transferred into a 96-well plate. The concentration of Evans Blue in the tissue was determined in duplicates by measuring the fluorescence intensity in a microplate reader (INFINITE 200 Pro, TECAN, Männedorf, Switzerland; excitation 620 nm, emission 680 nm) and calculated after linear regression analysis from a standard curve [50 (link)].