Xlumplanfl n 20x

Two-photon microscopy
was performed as described.^{38 (link)} Briefly,
surgical methods to access to the lung are based on Looney et al.^{79 (link)} Tail vein injection with BV421-labeled Ly6G
antibody (10 μg/mouse) (1A8, Biolegend) and 70 kDa tetramethylrhodamine-dextran
(200 μg/mice) (ThermoFisher Scientific) were performed to stain
PMN and lung microvascular structures, respectively, before surgery.
A resonant-scanning two photon microscope (Ultima Multiphoton Microscopes,
Bruker) with an Olympus XLUMPlanFL N 20x (NA 1.00) collected dual-color
images (Emission filter; 460/50 nm for Brilliant Violet 421 and 595/60
nm for tetramethylrhodamine) with 820 nm excitation at video rate.
Images were processed and analyzed by Image J and customized LabVIEW
programs. For PMN amount analysis, fluorescent intensities of PMN
in the field of view were quantified, and the value of saline injected
controls was normalized to 1. For PMN internalizing P/ANP, PMN number
with or without P/ANP in the field of view was counted and the percentage
was calculated. For PMN velocity analysis, PMN velocity of cells migrating
more than 1 min in the field of view was measured.

Mice bearing dorsal window chambers with MC38-mTAG-BFP2 cells were imaged to determine the kinetics of IL-12 induction in the tumor microenvironment. Dorsal window chambers were implanted into mice using well-established techniques 37 (link). Fluorescent tumor cells (MC38-mTAGBFP2) were implanted in the window chambers as previously described 47 ,48 (link) and allowed to grow for 8-10 days before imaging experiments, with tumor growth monitored regularly.
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). Tumor cells (MC38-TagBFP2), fusion-protein IL-12-GFP, and CANDI^AF647 were excited sequentially using a 405 nm, a 473 nm, and a 633 nm diode laser 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 tissue damage. Fiji (ImageJ, 2.3.0/1.54d) was used for image analysis.