Intravital Imaging: A Method for Cellular Level Observation of the Mouse Pancreas Through a Stabilized Imaging Window
Transcrição
Begin by switching on the intravital microscope, including the laser power. To insert a vascular catheter, apply pressure on the proximal side of the tail with the index and third finger. If necessary, heat the tail with a lamp. Disinfect the tail vein with a 70% ethanol spray, then, insert a 30 gauge catheter into the lateral tail vein and visualize the regurgitation of blood in the PE-10 tube. Apply silk tape on the catheter to stabilize it.
Inject FITC dextran and TMR dextran, or other fluorescent probes, as appropriate, according to the combination of fluorescent probes. Insert a rectal probe to automatically control the body temperature with the homeothermic heating pad system. Then, insert the pancreatic imaging window prepared during intravital microscopy setup into the window holder. Transfer the mouse from the surgical platform to the imaging stage.
To perform intravital imaging, start with imaging the pancreas at a low magnification, such as four times, to scan the whole view of the pancreas in the pancreatic imaging window. Once the region of interest has been determined, switch to a higher magnification objective lens, like 20 times or 40 times, to perform cellular level imaging with lateral and axial resolution approximately 0.5 micrometers and 3 micrometers, respectively.
Perform z-stack or time-lapse imaging to observe the 3D structure or cellular level dynamics, such as cell migration.
