<em>In Vivo</em> Imaging Uncovers the Migratory Behavior of Leukocytes within the Joints

Leukocytes are blood-borne cells derived from the bone marrow that must migrate into tissues to mediate inflammation. Controlling this migration, therefore, represents a critical point for the development of new therapeutic strategies to attenuate inflammation. Traditional endpoint studies have not allowed us to fully dissect the migratory behavior of leukocytes in vivo. Recent advances in imaging technology, however, have provided unprecedented insights into immune cell migration in live animals, greatly enhancing our understanding of the molecular regulation of immune cell trafficking. Here, we present a detailed protocol for in vivo imaging of the joints using a murine type II collagen-induced arthritis (CIA) model. Mice are immunized with type II collagen emulsified in complete Freund's adjuvant, and arthritis progression is monitored by clinical scoring and paw thickness measurements. At the peak of arthritis, leukocytes are visualized in vivo using confocal microscopy in transgenic mice expressing fluorescent proteins or in wild-type mice after intravenous injection of fluorescently labeled antibodies and high-molecular-weight Dextran for vessel staining. Careful surgical exposure of the joint, application of agarose, and proper positioning of the leg allow stable long-term imaging, typically lasting several hours. This approach enables real-time analysis of leukocyte arrest, crawling, and transendothelial migration within inflamed joints. This protocol will be valuable for research groups investigating leukocyte trafficking in arthritis and other inflammatory diseases. It may also serve as a platform for preclinical evaluation of therapies targeting immune cell migration.