Concanavalin a lectin cona

Quantification of leukostasis was performed at the end of the 8-week treatment by previously described method [27 (link)]. The chest cavity of each deeply anesthetized rat was carefully opened and a perfusion needle was inserted into the left ventricle. After cutting the right atrium, the animals were immediately perfused with 500 mL of PBS per kg body weight and heparin (0.1 mg/mL) to wash out nonadherent blood cells. Fluorescein isothiocyanate-coupled Concanavalin A lectin (ConA) (20 μg/mL in PBS; pH 7.4; 5 mg/kg; Vector Laboratories, Burlingame, CA, USA) was then perfused to label adherent leukocytes and vascular endothelial cells. Residual unbound ConA was flushed by PBS perfusion. Eyes were removed and fixed in 4% paraformaldehyde for 1 h. Retinas were dissected and flat mounted on a microscope slide, covered with anti-fading medium and a coverslip, and imaged via fluorescence microscopy. Only whole retinae in which the entire vascular network was stained were used for analysis. The total number of adherent leukocytes within the vessels of each retina was counted.

Young adult C57BL/6 mice received daily intraperitoneal injection of 75 mg/kg streptozotocin (STZ) for 5 consecutive days. Blood glucose levels were checked two days later and monitored weekly thereafter. Only animals with consistently elevated glucose levels over 350 mg/dL were used as diabetic mouse. Metformin was given daily after 5 days of STZ injection for 10 weeks. The retinal adherent leukocytes were quantified by staining adherent leukocytes in the vasculature of a flat-mounted retina as described previously [25 (link)]. Under deep anesthesia, mice were first perfused with 10 mL of PBS to remove erythrocytes and nonadherent leukocytes. This was followed by perfusion of 10 mL fluorescein-isothiocyanate (FITC)-coupled concanavalin A lectin (Con A; Vector, Burlingame, CA) to label the adherent leukocytes and endothelial cells, and then by another 10 mL of PBS to remove residual unbound lectin. Retina was removed, fixed in 1% paraformaldehyde, and flat-mounted. The total number of Con A-stained leukocytes adhering to the retinal vascular wall was counted under a fluorescence microscope as described above.