Intravital imaging of peritoneal macrophages through the intact abdominal wall. An intravital preparation for two-photon imaging through the intact abdominal is schematically depicted. Bhlhe40GFP mice were used to visualize peritoneal macrophages, as LPMs were high expressers of GFP in this mouse model, and B cells, another numerous leukocyte population in the steady state peritoneum, were GFP negative. GFP+ macrophages were observed between organs, which also showed some parenchymal cells with detectable GFP expression. Imaging in the steady state revealed nonadherent GFP+ macrophages flowing in the spaces between organs. Injection of Alexa Flour 594–conjugated heat-killed E. coli into the peritoneal space during imaging revealed that GFP+ macrophages accumulated in clusters on the mesothelial surfaces of organs. It appears as though bacteria floating by are captured by the adherent cluster of macrophages. More than three mice were imaged to draw conclusions about macrophage behavior in the steady state and after bacterial introduction. The frame rate is 23.98 frames per second.
