Intravital microscopy of leukocyte-endothelial cell interaction

Animals were anaesthetized by intraperitoneal injection of avertin 480 mg/kg, supplemented as necessary. Circulating leukocytes were fluorescently labeled by intravenous injection of 0.3 mg/kg rhodamine 6G (Sigma-Aldrich). The abdomen was opened in the midline, a loop of ileum exteriorized and the mouse placed on a heated stage (Harvard Apparatus WP-10). Second- or third-order mesenteric venules were observed by fluorescence microscopy using an Axio Observer D1 microscope with inverted configuration and a 20× objective, resulting in a final magnification of 200×. Images were acquired at 60 frames per second at high resolution by a digital video camera (Hamamatsu C11440-22CU CMOS Camera) and Zeiss software (ZEN Pro 2012). Rolling leukocytes was measured as the total number of leukocytes crossing a 100 μm venular segment in 60 seconds at a velocity significantly lower than the centerline velocity.