Fv1000mpe confocal imaging system

We used the dorsal skinfold chamber to image tumor cells in Arg1-eYFP mice. For MC38-H2B-mApple and EL4-H2B-mApple tumors, 10⁶ cells were injected in the fascia. Mice were monitored daily for 8 days prior to imaging. Where indicated, mice were injected i.v. with a Pacific Blue-labeled dextran nanoparticle (containing 1 nmol Pacific Blue dye) for TAM labeling or Pacific Blue-labeled 500 kDa amino-dextran (containing 56 nmol Pacific Blue dye) for vascular labeling 48 . Mice were anesthetized (2% isoflurane in oxygen) and immobilized on a custom heating platform to maintain body temperature and minimize motion artifacts. Imaging was performed with an Olympus FV1000MPE confocal imaging system and an XLUMPLFL 20x water immersion objective (NA 0.95; Olympus America). Pacific Blue, YFP and mApple were imaged sequentially using 405-nm, 473-nm and 559-nm light sources and BA430-455, BA490-540, and BA575-620 emission filters, along with DM405/473/559 nm dichroic beam splitters (Olympus America). Excised organs were also imaged on this microscope, but for these experiments Arg1-eYFP mice were injected 2.5 h prior with 75 μL of 10 kDa dextran-Cascade Blue (ThermoFisher), and 5 min prior with rhodamine-lectin (Vector Labs) for vascular labeling.