Concanavalin a lectin

Blood was removed from the vasculature of anesthetized animals by perfusion with phosphate-buffered saline (PBS; 10 mM phosphate buffer, 2.7 mM potassium chloride and 137 mM sodium chloride, pH 7.4; Sigma-Aldrich, St. Louis, MO) via a heart catheter. Animals were then perfused with fluorescein-coupled concanavalin A lectin (20 μg/ml in PBS; Vector Laboratories, Burlingame, CA), as described previously [20 (link),23 (link),26 (link)]. Flat-mounted retinas were imaged via fluorescence microscopy, and the number of leukocytes adherent to the vascular wall was counted.

Leucostasis was determined as previously described [30 (link)]. Briefly, at 2 months of diabetes, blood was removed from the vasculature of anaesthetised animals by extensive perfusion with PBS via a heart catheter. Subsequently animals were perfused with fluorescein coupled Concanavalin A lectin (20 μg/ml in PBS; Vector Laboratories, USA). Flat-mounted retinas were imaged via fluorescence microscopy and the number of leucocytes adherent to the vascular wall was counted.

Anesthetised mice were perfused with saline (154 mmol/l NaCl) followed by infusion of 10 ml of PBS containing 200 μl of fluorescein-coupled concanavalin A lectin (5 mg/ml; Vector Laboratories, Burlingame, CA, USA) [9 (link), 33 (link)]. Retinal flat-mounts were generated and brightly fluorescent leucocytes adherent to blood vessels were counted in the entire retina using fluorescence microscopy.

Retinal vasculature was stained and leukostasis was analyzed as previously described [15 (link),23 (link)]. Saline was perfused into the aorta to clear non-adherent leukocytes, then 10 mL of fluorescein-labeled concanavalin A lectin (1 mg/mL in PBS; Vector laboratories Burlington, ON Canada) was perfused to stain the retinal vasculature. After enucleation and isolation of the retina, flat mounts were imaged using a fluorescent stereoscope and the number of leukocytes adhered to the vasculature wall were counted (n = 3 samples/group). The 500 μm and 50 μm scale bar displayed gives a visual indicator of the size of the representative image.

Following a previously published method [33 (link), 36 (link)], rats were perfused via the right atrium with 0.1 M phosphate-buffered saline, pH 7.4, to clear blood cells and then rhodamine-conjugated Concanavalin A lectin (0.25 mg/kg, Vector Laboratories) to stain adherent leukocytes and the endothelium. Eyes were fixed in 4 % paraformaldehyde in 0.1 M phosphate-buffered saline, pH 7.4, for 30 min and retina flat-mounted. Non-overlapping images at ×200 magnification were captured, and the number of leukocytes per retina were counted. Six to nine rats per group were evaluated.