Abstract
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.
Highlights
Intravital microscopy of breast tumors in mice and rats at single-cell resolution[1,2,3,4] has resulted in important insights into mechanisms of metastatic behavior such as migration, invasion and intravasation of tumor cells[5, 6], angiogenesis[3] and immune cells response[7,8,9]
We have recently reported a technique to image orthotopic mammary carcinomas over multiple intravital imaging sessions in living mice[10]
We describe the protocol for the manufacturing of Mammary Imaging Window (MIW), insertion of the MIW on top of a tumor and imaging of the Dendra2- labeled tumor cells using a custom built imaging box
Summary
1. The area around the 4th nipple should have a small (5-7 mm diameter) tumor several days/weeks after injection of tumor cells depending on the cell type (Figure 1B). Put a piece of sterile gauze in the middle, and lay a few MIWs and sterile instruments around it: we use small standard scissors, spring scissors, microdissecting tweezers, microdissecting forceps, needle holder. 3. The mouse is anesthetized in the sterile hood using IP injection of 2.5% avertin (20 μl/g) in HBSS or, alternatively 10 μg/kg ketamine + 10 μg/g xylazine (contact your Animal Care Institute for approvals and ordering). 8. Transfer the animal onto a sterile surgical cloth inside the sterile hood. Place a hand heating pad (37 °C) (available in drugstores) on the bottom of the cage and cover it with gauze. The animal is allowed to recover over the 3-4 days before the first imaging session takes place
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