Quantifying EBD Extraction in Tissue

Eight rats were used for the in vivo experiments, which were performed under deep urethane anesthesia (4 g/kg, i.p.). Each rat was placed on a heating pad to maintain body temperature throughout the experiment. The right femoral vein and artery were cannulated. EBD, which was prepared as a 4% solution in 0.9% saline, was injected as a single bolus dose of 2 ml/kg via the venous cannula. After a 120 min period to enable uniform blood distribution, the EBD-stained blood was collected from the arterial cannula. The blood samples were placed on ice until centrifugation at 10,000 × g to sediment blood cells, and the EBD-stained plasma supernatants were collected in separate tubes. EBD extraction from plasma was accomplished via the addition of 50% TCA at 1:1-1:3 volume–ratios, which resulted in a [TCA_final] of 25.0, 33.3, and 37.5%, respectively. Immediately following blood collection, each rat was thoroughly perfused with 0.9% saline to rid the circulation of remaining dye, and the perfused brain and liver tissues were collected. For extraction, the brain and liver tissues were placed in 1:1–1:5 weight (mg):volume (µl) ratios of 50% TCA, and they were homogenized for 5 min (continuous beating) using a metal-bead homogenizer (BULLET BLENDER® BBX24). The TCA/extracts from the plasma, brain, and liver samples were centrifuged at 10,000 × g for 20 min to remove precipitates, tissue debris, and metal beads, and the supernatants were added to a 96-well plate (30 µl per well, each plate supplemented with 90 µl of 95% ethanol and thoroughly mixed by pipetting) for fluorescence spectroscopy (620 nm/680 nm).