Mice were anesthetized through an i.p. injection of Ketamine (80 mg/kg; Ceva, USA). Human IgE stimulation was performed by s.c. injecting human IgE mAb (60 ng/mouse; Calbiochem, USA) at the base of the ear. Twenty‐four hours after sensitization, mice were injected IV with 50 µL of 12.5 mg/mL Evans blue (Sigma–Aldrich). After 10 min, mice were i.p. injected with anti‐human IgE (50 µg in 100 µL/mouse; Dako, USA). After 15 min, dorsal skin and ears were collected. To stimulate MRGPRX2 receptor after Evans blue injection, 10 µL of a solution containing 1 µM of meglumine diatrizoate (Sigma–Aldrich), gadobutrol (Sigma–Aldrich), and (R)‐ZINC‐3573 (Sigma–Aldrich) were administered by intraplantar injection in one paw and the vehicle was administered in the other paw. Evans blue extravasation was visualized 15–20 min later and paws were collected. Samples were dried for 24 h at 55–60°C, weighed, Evans blue was extracted by a 24‐h incubation in formamide at 55–60°C and quantified by the absorbance at OD620nm (Tecan, USA).
Evans blue
Manufactured by Tecan
Sourced in Switzerland
Evans blue is a synthetic dye used in laboratory applications. It is a blue dye that binds to albumin in the bloodstream, allowing for the measurement of plasma volume and vascular permeability. The dye can be detected and quantified using spectrophotometric methods.
Automatically generated - may contain errors
Lab products found in correlation
Evans blue, by Merck Group (8 mentions)
Infinite m200 plate reader, by Tecan (2 mentions)
Infinite 200 pro, by Tecan (2 mentions)
Meglumine diatrizoate, by Merck Group (1 mentions)
Gadobutrol, by Merck Group (1 mentions)
R zinc 3573, by Merck Group (1 mentions)
Ketamine, by Ceva (1 mentions)
Formamide, by Merck Group (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Infinite m200 pro, by Tecan (1 mentions)
Trichloroacetic acid, by Merck Group (1 mentions)
Infinite m200, by Tecan (1 mentions)
Infinite 200 pro microplate reader, by Tecan (1 mentions)
8 protocols using evans blue
1
Evaluating IgE-Mediated Vascular Permeability
2
Quantifying Brain Vascular Permeability
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To quantify vascular and blood brain barrier repair, Evans blue (Sigma-Aldrich, Inc.) was injected retro-orbitally (2% w/v in saline, sterile filtered). Tissue was harvested after euthanasia and imaged to visualize topical Evans blue extravasation. Each brain was divided along midline to separate the left and right hemisphere and incubated in 500 μl Formamide at 55 degrees Celsius. After 24 hours, tissue debris was cleared by centrifugation (≥13,000 rcf, 15 minutes). Evans blue fluorescent was quantified by a TECAN infinite 200Pro plate-reader (excitation: 620 nm, emission: 680 nm). To account for gender differences, Evans blue fluorescence was normalized to express values as a function of body mass (n=6; 50:50 gender representation). Similarly, brain tissue was imaged on a Xenogen imager to qualitatively examine Evans blue epifluorescence in serial sections.
3
Evaluating Cerebral Vasculature Permeability
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To evaluate the permeability of the cerebral vasculature, we injected 2% Evans Blue (Sigma-Aldrich, Hamburg, Germany) tracer diluted in 0.9% NaCl (Fresenius Kabi, Bad Homburg, Germany; i.v.) 1 h after the induction of tMCAO. After 24 h, the animals were euthanized and brain tissue was removed. The tissue was cut in coronal sections (2 mm thick) that were incubated in 4% paraformaldehyde (Sigma-Aldrich, Hamburg, Germany) in the dark for 30 min and then divided into ipsi- and contralateral areas. Brain sections were weighed and incubated with formamide (Sigma-Aldrich, Hamburg, Germany) at 50 °C in the dark for the following 24 h. On the next day, samples were centrifuged for 20 min and 50 µL of the supernatant was collected and transferred into a 96-well plate. The concentration of Evans Blue in the tissue was determined in duplicates by measuring the fluorescence intensity in a microplate reader (INFINITE 200 Pro, TECAN, Männedorf, Switzerland; excitation 620 nm, emission 680 nm) and calculated after linear regression analysis from a standard curve [50 (link)].
4
Quantifying Blood-Brain Barrier Permeability
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At 2 h before the mice were anesthetized, 4 mL/kg of 2% Evans blue (Sigma) prepared with 0.9% NaCl was administered by tail vein injection. Then, they were perfused into heart with 0.9% NaCl. For quantitative measurement of Evans blue extravasation, the ischemic and nonischemic hemispheres were removed and homogenized in 1 mL of trichloroacetic acid and then centrifuged at 21,000 g for 20 minutes. Evans blue concentration was quantitatively determined by measuring the absorbance of the supernatant at 610 nm using spectrometer (infinite m200 pro, TECAN, Austria) [27 (link)].
5
Quantifying Vascular Permeability in EAE
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2% Evans Blue (Sigma-Aldrich) solution was intraperitoneally injected at onset of EAE disease. After 6 h, perfused spinal cords were weighed and homogenized in 1 ml 50% trichloroacetic acid (Sigma-Aldrich) and centrifuged for 10 min at 1000×g and 4 °C. Evans Blue concentration in the supernatant was measured on a plate reader (Infinite M200, Tecan Life Sciences) (excitation at 620; emission at 680) and quantified according to a freshly prepared standard curve.
6
Quantifying Vascular Permeability in Tick-Infested Mice
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To assess vascular permeability in WT and KO mice during tick bite, the mice were challenged with I. scapularis nymphs for 48 h. Then, 50 mg/kg Evans blue (Sigma, #E2129-10G) was injected intravenously into the WT (n = 6) and KO mice (n = 6). Any extravasated Evans blue present at the tick bite site was extracted in formamide and quantified spectrophotometrically at a wavelength of 610 nm using a Tecan infinite m200 plate reader (Tecan, Switzerland).
7
Quantifying Vascular Permeability in Tick-Infested Mice
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To assess vascular permeability in WT and KO mice during tick bite, the mice were challenged with I. scapularis nymphs for 48 h. Then, 50 mg/kg Evans blue (Sigma, #E2129-10G) was injected intravenously into the WT (n = 6) and KO mice (n = 6). Any extravasated Evans blue present at the tick bite site was extracted in formamide and quantified spectrophotometrically at a wavelength of 610 nm using a Tecan infinite m200 plate reader (Tecan, Switzerland).
8
Quantifying Tumor Vascular Permeability
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2×106 MGC803 cells in 0.2 mL PBS were subcutaneously injected into the right armpit region of BALB/c nude mice randomly divided into eight groups (n = 5 for each group). Two weeks later, PBS, CCL5 neutralization antibody (R&D Systems, MN), or TNF‐α neutralization antibody (R&D Systems, MN) were intratumorally injected every 2 days for 10 days. The mice were then injected intratumorally with 5% Evans Blue (Sigma, MA), then sacrificed 20 min later. Subcutaneous tumors were isolated, washed in PBS, then digested in 1 mL formamide at 60 °C for 48 h, while using 0.3 mL formamide to extract Evans Blue from the isolated axillary lymph nodes. A Tecan Infinite 200 Pro microplate reader (Tecan, Shanghai, China) was used to quantify the amount of extravasated Evans Blue dye at 630 nm.
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