Texas red 70 kda dextran

Imaging was performed on an FV1000MPE multiphoton laser scanning microscope (Olympus Corp., Japan) with an InSight DS tunable laser (Spectra-Physics, USA). A 25× water-immersion objective lens (XLPN25XWMP2, NA 1.05, WD 2 mm, Olympus Corp., Japan) was co-aligned with the ring transducer and water-coupled for imaging at lateral and axial resolutions of 0.994 μm/pixel and 2 μm/slice, respectively, and at an imaging speed of 2-8 μs/pixel for a maximum imaging duration of 4 h. Texas Red 70 kDa dextran (dissolved in PBS, 5 mg/kg; Invitrogen, Canada) was injected through a tail vein catheter for visualization of vasculature. EGFP cells and Texas Red dextran were excited at 900 nm. Fluorescent emissions were collected with photomultiplier tubes preceded by the following bandpass filters: 575-645 nm for Texas Red and 495-540 nm for EGFP.
After administration of Texas Red dextran, XYZT image stacks (XY: 0.994 μm/pixel, typically 512 × 512 pixels; Z: 2 μm/slice, 6-10 slices) were acquired to assess baseline immune cell dynamics. Regions-of-interest (ROIs) were centred on blood vessels that showed increased permeability following FUS+MB treatment. Imaging duration ranged from 2-4 h and was limited by increased noise from fluorescent cells reacting to the acute cranial window in the superficial layers of the cortex.

Multiphoton imaging of mammary tumors was done as described previously [56] . MDA-MB-231 cells labeled with Dendra2- or cortactin-GFP were injected into the mammary fat pads of 5–7 week old SCID mice. After 10–12 weeks, we injected Texas Red 70 kDa dextran (250 nmol in 100 µl PBS per injection) [5] (link) for macrophage labeling and performed skin flap surgery 2 h later on anesthetized animals. Exposed mammary tumors were positioned on top of a coverslip on an inverted microscope and imaged continuously using a custom-built two-laser multiphoton microscope for up to 3–5 h. In some experiments, additional tail-vein injections were done for blood vessel labeling using Texas Red 70 kDa dextran (Invitrogen; 250 nmol in 100 µl PBS per injection), MMPSense 680 (Perkin Elmer; 2 nmol in 150 µl PBS per injection), SR-FLIVO (Immunochemistry Technologies; 1∶10 dilution, 2 h prior to imaging), or the pan-MMP inhibitor GM6001 (Milipore; 1 µmol in 100 µl injection). A stock solution of GM6001, 500 mM in DMSO, was diluted in sterile PBS before tail vein injection. Post-surgical injection assures that compounds are only present in tumor blood vessels that are intact, connected to tumor vasculature and flowing at the time of injection.