Abstract
Actin plays a crucial role during cell motility, but the organization of F-actin filaments during lymphocyte migration has not been visualized in vivo. Here, we present a 4D imaging platform using high-resolution confocal intravital microscopy to precisely determine the F-actin filament profile during lymphocyte transendothelial migration and interstitial migration. This protocol allows prolonged live imaging by laser scanning microscopy with advanced spatial resolution compared with the traditional multi-photon intravital microscopy techniques. For complete details on the use and execution of this protocol, please refer to Yan et al. (2019).