Quantifying Retinal Leukocyte Adhesion

Using an established method, rats were perfused via the right atrium with 0.1M PBS, pH 7.4 to remove non-adherent blood cells, followed by rhodamine-conjugated Concanavalin A lectin (0.5 mg/kg, cat# RL1002, Vector Laboratories, Inc., CA, USA) to stain the vasculature and adherent leukocytes [13 (link), 29 (link)]. Eyes were fixed in 4% paraformaldehyde in PBS for 30 min and retina flat-mounted on microscope slides. The vasculature was observed using an Olympus BX51 fluorescent microscope (Olympus, Tokyo, Japan). Non-overlapping images were captured and the number of leukocytes per retina counted at 200x magnification. Five to six rats per group were evaluated.