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In the last decade, intravital microscopy of breast tumors in mice and rats at single-cell resolution1-4 has resulted in important insights into mechanisms of metastatic behavior such as migration, invasion and intravasation of tumor cells5, 6, angiogenesis3 and immune cells response7-9. We have recently reported a technique to image orthotopic mammary carcinomas over multiple intravital imaging sessions in living mice10. For this, we have developed a Mammary Imaging Window (MIW) and optimized imaging parameters for Dendra211 photoswitching and imaging in vivo. Here, we describe the protocol for the manufacturing of MIW, insertion of the MIW on top of a tumor and imaging of the Dendra2- labeled tumor cells using a custom built imaging box. This protocol can be used to image the metastatic behavior of tumor cells in distinct microenvironments in tumors and allows for long term imaging of blood vessels, tumor cells and host cells.
In order to fabricate the plastic base, we are currently using silicone rubber casting molds created using hand-made MIWs. The mold is comprised of two parts of silicone rubber that make an exact replica of both the front and back of the original when filled with polyester resin and then immediately joined together. The liquid polyester resin is mixed together 9:10 and results in a hard structure when fully cured in 48h. The plastic is UV protected and archival without breaking down or becoming yellow over time. After the base is cured, steps 2.8-2.16 are done the same way as for manually-made MIWs.
The imaging box assures that the MIW is sitting flat, right above the microscope objective. It also facilitates temperature and anesthesia control through constant airflow of isoflurane. The box is made of plexiglass and glued together using plastic weld. It is fitted for the specific microscope stage and therefore the shape of its bottom varies depending on the microscope used (Figure 2 shows the scheme of the box fitted to Leica SP5 microscope stage). Dimensions of the box in centimeters are l=11.4 cm, w=7.6 cm, h=4.4 cm (Figure 2A). The bottom of the box consists of a hollow plate, 12.7x8.4 cm in size, which fits inside the Leica SP5 microscope stage, and two sliding “doors”, 5.7 X 5.7 cm_each, which form a circular opening (d=2.22 cm) when closed. The MIW fits into this opening. The front piece contains the inlet hole for the anesthesia delivery while one of the side pieces contains an outlet hole which leads to the vacuum. Note that the condenser and the slide holder need to be removed prior to the imaging box placement.
The procedure is described for the Leica SP5 confocal microscope; the laser power at the focal plane, available objectives and software options vary when using other confocal microscopes or multiphoton imaging set up, and therefore the protocol described below should be used as a guide to optimize the experiment on your microscope.
Figure 3C shows a rectangular photoswitched region (red) oriented orthogonally relative to the blood vessel (no fluorescence). Non-photoswitched cells are green, while the scattering from the extracellular matrix is purple. Image is a maximum intensity projection of four images along the Z-axis (20-50 μm depth).
This work was supported by US Department of Defense (BC075554 to B.G. and BC061403 to D.K.), US National Institutes of Health (U54GM064346 to J.v.R.; CA100324 to J.C., J.E.S. and J.W.; CA77522 to J.E.S., U54CA126511 to J.C. and B.G.). We thank D. Entenberg for help with microscopy, M. Rottenkolber for help in fabrication of the imaging box and J. Pollard (Albert Einstein College of Medicine) for the F4/80 antibody.