Evans blue dye leakage

Evans blue dye (2% in saline) was administered intraperitoneally (80μL) and animals were sacrificed and perfused 2 hours after injection. Photographs of brain tissue were taken immediately after tissue isolation. Evans blue was extracted by homogenizing brain tissue in 50% trichloroacetic acid (wt/vol) solution, and centrifuged at 16,000 g for 20 minutes. The supernatants were diluted with ethanol (1:3), and fluorescence was quantified by using a microplate fluorescence reader. Sample value was determined by using a standard curve of Evans blue (0.1 to 100 ng/ml in the same solvent). Result was shown as nanograms of Evans blue per milligram of tissue⁴⁷.