Fitc conjugated dextran

To detect permeability in 2-week old mice, the abdomen was opened and a ligation was performed in the ileum at 2 mm above the cecum. FITC-conjugated dextran dissolved in water (4000 mol wt, Sigma-Aldrich) was injected into the ileum at 4 mg/10 g bodyweight. Then the abdomen was closed. In adult mice with DSS treatment, colitis occurred in the colon. A ligation was performed in the colon at 2 mm below the cecum, then FITC-dextran was injected into the colon. Whole blood was collected using heparinized micro-hematocrit capillary tubes via eye bleed 2 h after FITC-dextran administration. Fluorescence intensity in sera was analyzed using a plate reader. The concentration of FITC-dextran in sera was determined by comparing to the FITC-dextran standard curve.

Window assays were performed as described previously [50 (link), 51 (link)]. Briefly, a 5 mm diameter flap of skin was dissected away from the dorsal skin flap of anesthetized recipient wild-type or EphA2-deficient three month old Balb/c female mice, leaving a fascial plane with associated vasculature. A gelfoam sponge (approximately 1 mm in diameter)impregnated with 1 μg of Slit2, Robo1-Fc, or control IgG in 50% Matrigel/PBS was implanted in the window chamber adjacent to a portion of 4T1 tumor (approximately 0.7 mm in diameter) isolated from a donor mouse. The chambers were sealed with glass coverslips and photographed on 1 day following implantation to measure initial tumor size and baseline vascular morphology. 7 days after implantation, FITC-conjugated dextran (2% in PBS, Sigma-Aldrich) was injected intravenously, and tumors in window chambers were photodocumented using an Olympus BX60 microscope and digital camera. Branches from host blood vessels within the window chambers were enumerated in at least three independent fields per mouse, and statistical significance was determined by two-tailed, paired Student’s t-test. Data are a representation of 6–8 independent samples per condition with standard error of the mean, and statistical significance was assessed by two-tailed, paired Student’s t-test.

To measure intestinal permeability in the present mouse model of opioid dependence, FITC-conjugated dextran (Sigma-Aldrich, St. Louis, MO) or PBS was used as described previously (Kang et al., 2017 (link)). Following naloxone-precipitated withdrawal, mice received oral gavage of FITC-labeled dextran (44 mg/100 g body weight). After 4 h, whole blood was collected, and plasma was isolated by centrifugation for 15 min at 3000 rpm and 4 °C. FITC concentration was fluorometrically quantified by emission spectrometry (Promega, Madison, WI) at 528 nm, using an excitation wavelength of 485 nm. All concentrations were measured against a standard curve of serially diluted FITC-dextran.