Anti–JAM-C mAb suppresses TNFα-stimulated monocyte accumulation in the mouse cremaster muscle tissue. (A) CX₃CR1 ^GFP/GFP mice were injected intravenously with an isotype control mAb or the anti–JAM-C mAb H33 (both at 3 mg/kg), and 15 minutes later, mice were injected with intrascrotal TNFα (300 ng/mouse). After 2 hours, the cremaster muscle was exteriorized, and leukocyte transmigration was quantified for 2 hours at different time points by intravital microscopy. Error bars represent SEM. Results are from n = 3 mice/group, and statistical comparisons are shown by lines (*P < .05). (B) A representative micrograph of a TNFα-stimulated cremasteric venule. The fixed section was immunostained with an anti–α-SMA (SMC/pericyte marker) to visualize the venular wall (red) and contained GFP-labeled monocytes (green), reconstructed in 3D and captured by still confocal microscopy (“Intravital microscopy” and “Flow assays”) in order to optimize resolution. The image shows monocytes both in the luminal compartment and in the extravascular tissue. Scale bar equals 10 μm. (C) There was a reduction in monocytes entering the extravasculature of mice treated with blocking antibody to JAM-C. At the end of the intravital microscopy studies detailed, cremasteric tissues were dissected away from the mice and analyzed for monocyte numbers (visualized by their GFP label) in the extravascular tissue by confocal microscopy. Data show that mice treated with the anti–JAM-C mAb exhibited a significant reduction in abluminal monocyte numbers (*P < .05). Error bars represent SEM.