Ba490 540

Intravital microscopy was performed on an Olympus FV1000 confocal-multiphoton imaging system using a XLUMPLFLN 20x water immersion objective (NA 1.0; Olympus America) with 2x digital zoom. Images were scanned sequentially using 405-nm, 473-nm, 559-nm, and 635-nm diode lasers with a DM405/473/559/635-nm dichroic beam splitter; emitted light was collected using combinations of beam splitters (SDM473, SDM560, and/or SDM 640) and emission filters BA430-455, BA490-540, BA575-620, and BA655-755 (all Olympus America).
Dorsal window chamber imaging was performed following previously described procedures (16 (link)); briefly, 2 million HT1080-membrane-mApple cells in 50 μl PBS were injected under the fascia of nu/nu mice (Cox7, MGH) 30 min after 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.).

Dorsal windows were implanted into IL-12 eYFP reporter mice.^{53 (link), 54 (link)} All confocal images were collected using a customized Olympus FV1000 confocal microscope (Olympus America). A 2x (XLFluor, NA 0.14), a 4x (UPlanSApo, NA 0.16), and an XLUMPlanFL N 20x (NA 1.0) water immersion objective were used for imaging (Olympus America). MC38 H2B-apple tumor cells, HAMT^AF647, and vascular probes were excited sequentially using a 405 nm, a 473 nm, a 559 nm, and a 633 nm diode laser, respectively, in combination with a DM-405/488/559/635 nm dichroic beam splitter. Emitted light was further separated by beam splitters (SDM-473, SDM-560, and SDM-640) and emission filters BA430-455, BA490-540, BA575-620, and BA655-755 (Olympus America). Confocal laser power settings were carefully optimized to avoid photobleaching, phototoxicity, or damage to the tissues. FIJI (ImageJ, 2.9.0/1.53t) was used for image analysis. HAMT^AF647 was administered as a single injection containing a mixture of HAMT 1+2+3 (5 mg per injection, 100 μL) and CANDI^AF647 (5 mg, 100 μL).

Non-stressed mice versus mice restraint-stressed for 1 h for neutrophil imaging, as well as non-stressed mouse versus mice restraint-stressed for 4 h for B cell, T cell and monocyte imaging, were anaesthetized with 1–3% isoflurane and 2 l min⁻¹ oxygen anaesthesia. Mice were kept on a 37 °C heating plate during the whole procedure. The fluorescence agents FITC-dextran (2 × 10⁶ Da) for labelling the vasculature and PE-labelled antibodies to visualize different cell types (5 μl antibody stock in 50 μl PBS) were injected intravenously and one mouse ear was immobilized on a 37 °C heated aluminium plate for confocal imaging. Without delay, the mice were imaged under isoflurane anaesthesia. The whole imaging procedure was kept to a maximum of 1 h. Imaging was performed using 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). Neutrophils were labelled using PE-labelled anti-Ly-6G antibodies; B cells with PE-labelled anti-CD19 antibodies; T cells with PE-labelled anti-CD3 antibodies; and monocytes with PE-labelled anti-CD115 antibodies.