Real-time Imaging of Myeloid Cells Dynamics in Apc^Min/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

The overall goal of this procedure is to visualize myeloid cell dynamics within the intestinal tumors of a live animal. This is accomplished by first injecting fluorescent molecules and atropine in a PC min mice. In the second step, the intestine is prepared for imaging and the mouse is positioned on the microscope stage.

In the final step, images of the tumor within the live animal are acquired, ultimately spinning disc confocal. Microscopy can be used to visualize the behavior of myeloid cells within the tumors of live a PC min mice over several hours. This method can help answer key questions in the cancer field, such as what is the role of human cells in colorectal cancer?

Begin by using lab tape to secure two cover slips onto the stage insert of the microscope imaging platform. Then place the insert onto the stage and clean it with alcohol wipes. Next, turn the hot bead sterilizer to 250 degrees Celsius, and then sterilize the surgical instruments for 30 seconds while the tools are cooling.

Place this styrofoam lid in between two lab soakers on the surgery bench. Then when the mouse is breathing deeply and slowly, place the animal on the surgical stage on its flank and retroorbital. Inject the DExT strand, rod Domine, and GR one antibody solution.

After the injection, turn the mouse onto its back with its nose in the anesthesia cone, and use an electric shaver to remove the hair from the ventral surface and the left hind limb of the animal. When the fur has been removed, place a new lab soaker onto the surgical stage to avoid hair contamination, and then disinfect the ventral surface of the animal with alcohol wipes and Betadine. Next subcutaneously, inject the atropine solution into one flank of the mouse, and then use scissors and a pair of forceps to make a one centimeter ventral midline incision through the skin and the peritoneum.

Carefully pull out three to four centimeters of intestine and identify one or several tumors to image. Then position the loop of the intestine containing the tumor of interest on top of a glass microscope slot. Place a few drops of super glue along the intestine to attach it to the slide after the glue has dried for a minute, use a marker to draw a line on the slide pointing to the tumor to facilitate its localization during imaging.

Now transfer the mouse carefully onto the microscope stage with its ventral side down and its nose in the nose cone. Secure the anesthesia line with lab tape and then reposition the setup so that the intestine is in the center of one of the cover slipped windows. Gently tape down the slide and attach the oximeter probe to the left limb of the mouse to follow its heart and respiratory rates and the arterial oxygen saturation levels.

Then cover the mouse with the heating blanket to avoid hypothermia and decrease the ISO fluorine concentration for imaging. Finally, in the multidimensional acquisition window, click acquire to start the acquisition. When the acquisition is finished, the movie is recorded and saved by using a Mars software.

By using spinning disc confocal microscopy, non-tumor, and tumoral tissues. In the small intestine of a PC min mice can be visualized from the serosal surface after intravenous injection of fluorescent dextran rodine and a lymphocyte antigen six complex locus G 6 47 conjugated antibody, blood vessels and polymorphonuclear cells can be detected respectively. PIs patches that are aggregations of lymphoid tissue and full of myeloid cells can also be easily observed.

The Crips of the small intestine are surrounded by blood vessels and myeloid cells. Tumoral tissue is often more vascularized and more infiltrated by myeloid cells, and the structure of the Crips is less well organized depending on the size of the tumor. The expression of actin ECFP by the smooth muscle layer can sometimes make a sharp focus of the intestinal epithelium difficult to achieve as can be observed in this image, myeloid cell dynamics in tumors can be followed in real time for a few hours as demonstrated in this movie, whereas G one positive neutrophils circulate in blood vessels and patrol the tissues.

Other myeloid cells, mostly macrophages, surround the Crips and are less modal. After watching this video, you should have a good understanding of how to visualize myeloid cell dynamics within intestinal tumors in live animals.