Evans blue

Mice were injected intravenously (iv) with 0.2 ml of 1–2% Evans Blue (Sigma). Mice were sacrificed 1 h later, and tumors were weighed and placed in formamide (2 ml) (Merck) (37 °C, 48 h) to extract Evans Blue dye from the tissue. Absorbance was measured at λ = 620 nm (Bio-Rad SmartSpec 3000). Evans Blue concentration was calculated from a standard curve and is expressed as µg of Evans Blue per g of tumor tissue.

BBB disruption was assessed by measuring the amount of Evans blue extravasation. Briefly, mice were intravenously injected with 0.1 ml 4% Evans blue (Sigma‐Aldrich). After 60 min, the animals were perfused transcardially with phosphate‐buffered saline (PBS) to remove intravascular Evans Blue. The brains were homogenized in formamide (Sigma‐Aldrich) and incubated in the dark at 60°C for 24 h. The absorbance of eluted Evans blue dye in formamide solution was measured using a spectrophotometer at 610 nm and normalized to plasma levels.

To measure the BRB and BBB integrity during the periods of microglial depletion and repopulation, 2-month old male mice were fed by PLX5622 or CD for 14 days, and PLX5622 for 14 days followed by CD for 7 days. Mice were then intraperitoneally injected with 0.2 mL 2% Evans blue (Sigma). Six hours after injection, mice were systematically perfused by 0.9% normal saline and tissues were then harvested. Tissue homogenate was incubated with formamide (1 g tissue in 10 mL formamide, Sigma) at 55 °C for 24 h to extract Evans blue. After that, the formamide-Evans blue mixture was centrifuged and the supernatant absorbance was measured at 610 nm by a microplate reader.

Both flanks of adult anaesthetized mice were shaven. The next day, 100 µl of 1% Evans Blue (Sigma-Aldrich) in sterile saline (wt/vol) was injected intravenously through the lateral tail vein. In some experiments, mice received an intraperitoneal injection of pyrilamine maleate (4 mg/kg body weight in 0.9% saline; Sigma-Aldrich) before Evans Blue injection to inhibit release of endogenous histamine. 30 min after Evans Blue injection, 20 µl of PBS or PBS containing 50 ng VEGF164 (PeproTech), 300 ng SEMA3A (R&D Systems), or 50 ng histamine (Sigma-Aldrich) were injected intradermally each at three sites into the flank skin of anaesthetized mice. After 20 min, mice were culled, the skin was separated from the underlying muscle, and the tissue surrounding the injection sites excised (circled in Fig. 1 A) and dried overnight at 55°C. Evans Blue was extracted by incubation in formamide at 55°C overnight and quantified by spectrophotometry at 620 nm after subtraction of background absorbance at 740 nm. Data from the three sites injected with the same agent (ligand or PBS) were averaged and expressed as fold change relative to PBS control per each mouse. In some experiments, the inner side of the skin was imaged on an MZ16 stereomicroscope (Leica) equipped with a Micropublisher camera (Perkin-Elmer). In other experiments, a sample of liver or skin tissue was retained for immunoblotting.

The vessel permeability assay was performed as described previously [36 (link)]. Evans blue solution (0.5% Evans blue (Sigma-Aldrich, St. Louis, USA) in PBS) was prepared and filtered through 0.22 μm filter. The Evans blue solution was advanced into the lateral tail vein towards the direction of the head in mice for 60 minutes. The liver and heart were collected and immersed in 4% paraformaldehyde for 24 hours at 55°C before analyzing the absorbance of Evans blue (610 nm) by using a multi-well scanning spectrophotometer (Thermo, Multiskan, Spectrum).

In addition to FITC-dextran, we used the Evans blue dye assay to determine the permeability of the BBB [69 (link)]. Briefly, rats received 4% of Evans blue (Sigma, St. Louis, MO, USA) through the intraperitoneal route (4 mL/kg). After 6 h post-administration, rats were perfused transcardially with PBS (Sigma, St. Louis, MO, USA) followed by the harvesting and weighing of brain tissues. Evans blue in brain tissue was extracted by homogenizing the samples in 0.1 mol/L PBS (pH 7.4), followed by protein precipitation using 60% trichloroacetic acid (Sigma, St. Louis, MO, USA). The samples were then vortexed and cooled. After 30 min, the samples were centrifuged and the concentration of Evans blue in the supernatant was determined at a wavelength of 610 nm using a spectrophotometer. A standard curve was plotted using Evans blue dye and the results were expressed as µg/g brain tissue.