Evans Blue

Evans Blue (Direct Blue 53; T-1824; C.I. 23860) is a potent inhibitor of L-glutamate uptake via the membrane bound excitatory amino acid transporter (EAAT). Evans Blue is a L-glutamate and kainate receptor-mediated currents inhibitor. Evans Blue has a strong affinity towards serum albumin, making it a high molecular weight protein tracer. Evans Blue is also used to study BBB (blood-brain barrier) permeability^{[1][2][3][4][5]}. In Vitro:Guidelines (The following is our recommended protocol, which serves only as a guideline and should be modified according to your specific needs).
(I) Preparation of stock solution Weigh
0.05 g of Evans Blue powder and dissolve it in 10 mL of DPBS (without Ca²⁺/Mg²⁺). After complete dissolution, filter through a 0.22 μm filter for sterilization and store at room temperature away from light.
(Ⅱ) Live Cell Staining
1. Transfer 100 μL of cell suspension to a centrifuge tube, add 100 μL of 0.5% Evans Blue, and mix gently.
2. Stain at room temperature (the duration can be appropriately extended, but not exceeding 10 min).
3. Take a small amount of cells, drop them onto a hemocytometer, and count blue cells (dead cells) and total cells under a microscope.
4. Cell viability = (Total number of cells − Number of blue cells) / Total number of cells × 100%.
0.5% Evans Blue Staining Solution accurately distinguishes between dead and live plant cells via selective staining of membrane-damaged cells^[1]. In Vivo:Guidelines (The following is our recommended protocol, which serves only as a guideline and should be modified according to your specific needs).
(I) Preparation of stock solution Weigh
0.05 g of Evans Blue powder and dissolve it in 10 mL of DPBS (without Ca²⁺/Mg²⁺). After complete dissolution, filter through a 0.22 μm filter for sterilization and store at room temperature away from light.
(II) Blood-Brain Barrier Permeability Assay
1. Inject 200 μL of 0.5% Evans Blue solution via tail vein into mice (reference dose for 25-35 g mice: approximately 5.7-8 μL/g; adjust the dose for other animals based on body weight ratio).
2. Confirm successful injection by observing the blue discoloration of the mice's eyes and skin.
3. Sacrifice the animals 0.5-1 h later, collect the target brain tissue, homogenize it, and centrifuge to obtain the supernatant.
4. Protein precipitation (choose one of the two methods):
Mix the supernatant with an equal volume of trichloroacetic acid and incubate at 4°C;
Mix the supernatant and acetone at a ratio of 3:7, then incubate at room temperature for 24 h.
5. Centrifuge for 15 min, then collect the supernatant to measure absorbance at 620 nm.
6. Simultaneously measure the gradient of Evans Blue standards, plot a standard curve, and calculate the sample content.
(Ⅲ) Pulmonary Vascular / Epithelial Permeability Assay
1. Inject 200 μL of 0.5% Evans Blue solution via tail vein into mice and allow circulation for 1 h.
2. Sacrifice the mice, collect blood, and centrifuge to separate serum.
3. Perform bronchoalveolar lavage (BAL) via tracheal intubation and collect the lavage fluid.
4. Perfuse the right ventricle with DPBS, collect lung tissue, and extract with formamide at 55°C for 48 h.
5. Measure the absorbance of diluted serum (1:10), undiluted BAL fluid, and lung extract at 620 nm / 740 nm respectively, then quantify using the standard curve after turbidity correction.
6. Normalization: The ratio of BAL fluid concentration to serum concentration reflects epithelial barrier function; the ratio of lung tissue concentration to lung weight to serum concentration reflects endothelial barrier function.
0.5% Evans Blue Staining Solution identifies degenerative and apoptotic muscle fibers in mdx mice with muscular dystrophy^[1].
0.5% Evans Blue Staining Solution identifies necrotic cardiomyocytes in rats with myocardial cell injury induced by myocardial contrast echocardiography^[1].
0.5% Evans Blue Staining Solution (administered for 48 h) specifically and persistently labels necrotic liver tissues in brown rats (rats) with reperfusion-induced partial hepatic infarction^[1].
0.5% Evans Blue Staining Solution accurately delineates the core region of myocardial infarction in rabbits with reperfused myocardial infarction^[1].
0.5% Evans Blue Staining Solution (administered subcutaneously) effectively localizes lymphatic drainage pathways and identifies draining lymph nodes in normal mice^[1].
0.2% Evans Blue Staining Solution accumulation in the brain of mice following collagenase-induced intracerebral hemorrhage is independent of the administration route^[2].