Sdm560

Intravital microscopy was performed in dorsal skin-fold window chambers installed on DPE-GFP or GREAT mice inoculated with MC38-H2B-mApple tumors. Mouse macrophages and/or vasculature were labeled with Pacific Blue ferumoxytol and dextran, respectively. AF647-aPD-1 (200 μg) was delivered i.v. and its tumor distribution was observed using an Olympus FluoView FV1000MPE confocal imaging system (Olympus America), as described previously (44 (link)). Pacific Blue, GFP/YFP, mApple, and AF647 were imaged sequentially using 405, 473, 559, and 635 nm lasers and BA430-455, BA490-540, BA575-620, BA575-675 emission filters with DM473, SDM560, and SDM 640 beam splitters, all sourced from Olympus America. Time lapse images were acquired continually over the first hour following AF647-aPD-1 injection, after which the mice were allowed to recover before subsequent imaging.

TNPs were injected at the indicated dose (1 mg kg⁻¹ unless otherwise stated) via tail-vein catheter immediately after mixing to a final 1x phosphate-buffered saline (PBS) solution, at a final volume of 100 μl. Intravital microscopy was performed on an Olympus FV1000 multiphoton imaging system using a XLUMPLFLN 20x water immersion objective (NA 1.0; Olympus America). Images were scanned sequentially using 405-nm, 473-nm, 559-nm and 633-nm diode lasers in combination with a DM405/488/559/635-nm dichroic beam splitter. Emitted light was then separated and collected using appropriate combinations of beam splitters (SDM473, SDM560 and/or SDM 640) and emission filters BA490-540, BA575-620, BA575-675 and/or BA655-755 (all Olympus America). Dextran pacific blue (λ_ex = 405 nm) was injected to initially image TMV as previously described^{28 (link)}. Briefly, 500 kDa amino-dextran (Thermo) was labeled with Pacific Blue succinimidyl ester (Thermo), purified using 30 kDa MWCO centrifugal filtration (Amicon), and 250 μg intravenously injected 10 min prior to imaging. Dorsal window chamber imaging was performed following previously described procedures^{28 (link),67 ,68 (link)}. Briefly, 2 million HT1080-53BP1-mApple cells were suspended in 50 μl PBS and injected under the fascia of nu/nu mice (Cox7, MGH) 30 min following surgical chamber implantation, and imaged two weeks later.

Fed mice versus fasted mice were anesthetized with 1–3% isoflurane and 2 l/min oxygen anesthesia for monocyte imaging. Mice were kept on a 37°C heating plate during the whole procedure. Fluorescent agents, FITC dextran (2 million Da) for labelling the vasculature and PE-labeled anti-CD115 antibodies to visualize Ly-6C^hi monocytes (5 μl antibody stock in 50 μl PBS), were injected i.v. The femoral vein was exposed on a 37°C heated plate and the vessel lumen was imaged confocally. The entire surgical and imaging procedure was kept to a maximum of 1h. Imaging was done with an Olympus XLUMPLFLN 20X W NA:1.00 water immersion objective on an Olympus custom made confocal multi-photon microscope using 473 nm and 559 nm diode-lasers with a DM405/473/559 dichroic mirror, a SDM560 beam splitter, and BA490–540 and BA575–675 emission filters (Olympus America Inc.).