Extravasation assay of Evans blue dye

We calculated capillary permeability of lungs in animals by Evans blue dye extravasation, by a method previously published [20]. We introduced 30 mg/kg Evans blue dye into the internal jugular vein of animals 1 h prior to euthanasia. During the experiment, we collected heparinized blood through cardiac puncture, followed by PBS perfusion of the right ventricle to eliminate pulmonary intravascular dye. Lung tissue was isolated and pulverized in PBS. We extracted Evans blue using 2 volumes of formamide (Sigma-Aldrich) followed by overnight incubation at 60°C, followed by centrifuging at 5000 g for 30 min at 4°C. The absorption of Evans blue dye in the lung supernatants and plasma was measured at 620 nm and corrected for heme component as follows: A₆₂₀ (corrected)=A₆₂₀−(1.426×A₇₄₀+0.030). We thus calculated the Evans blue index as the ratio of the amount of dye in the lungs to the concentration of plasma dye.