Dendra2 Photoswitching through the Mammary Imaging Window

This video illustrates a protocol beginning with the injection of a fluorescent protein expressing tumor cell line into the mouse mammary fat pad. A specialized chamber for in vivo imaging is constructed and then surgically implanted into the animal at which point imaging data can be acquired. Hi, I'm Dr.Vic from the laboratory of Dr.John Condi and Dr.Jeffrey Siegel of Biophotonics Center at Albert Einstein College of Medicine.

Today we will show you a procedure for intravital photo switching and imaging of DRA two labeled cancer cells through the memory imaging window. We use this procedure in our laboratory to study influence of the tumor microenvironment on the motility of tumor cells. So let's do it Wearing latex gloves.

Begin the fabrication of the mammary imaging window, or MIW by heating up a five to 10 centimeter tissue culture dish and creating a curved surface by pushing a rounded hard object such as a one inch Dremel bit against the dish. Cut out the center of the dish using a heated razor blade. Now use a small cone-shaped dremmel bit to make a six to seven millimeter diameter hole in the center of the dome shaped plastic base.

Make the edges of the plastic base completely smooth by sanding it with the Dremel and further filing it. File the top of the plastic base to make a flat surface for the glass cover slip. The diameter of the filed flat surface should be nine to 10 millimeters.

Next, glue the eight millimeter circular glass cover. Slip onto the flattened surface using super glue and let it dry for 15 minutes. Using a heated 26 G needle, make eight suturing holes by puncturing from the outside to the inside of the base, the holes should be evenly distributed around the cover.

Slip nwt 0.5 to one millimeter away from the edge of the cover slip. Wash the MIW with deionized water, and then again with 70%ethanol. Use a Q-tip to clean the glass until it is completely transparent.

Use acetone to remove any remaining foggy spots from the glue vapor. Finally sterilize the MIW by exposing it to ultra filet light for three hours on each side. Before inserting the MIW, the area around the fourth nipple should have developed a small five to seven millimeter diameter tumor as a result of the injection of tumor cells.

In part one Check to make sure that the tumor is not visibly necrotic. There should be intact skin with hair on the area around it. To begin, prepare a sterile space for the surgery by laying down a piece of sterile cloth inside a sterile hood.

Put a piece of sterile gauze in the middle and gather the items you will need for the surgery. You'll need a pair of small and a pair of large dissection, spring scissors, micro dissecting tweezers and forceps, and a needle holder After anesthetizing the mouse with 2.5%averting, use a small animal shaver to shave the area above the tumor. Use their hair removal cream to remove the rest of the hair and clean the skin.

Using ethanol dipped Q-tips. Transfer the animal onto the sterile gauze and apply ophthalmic ointment to the eyes to keep them from drying out and becoming infected. Sterilize the skin with Betadine and then clean it with 70%ethanol.

With the annual and sterile surgical cloth, pull the skin immediately medial to the nipple using forceps and cut an approximately two millimeter incision. Separate the underlying mammary fat pad from the skin using dissection, scissors, and forceps. When separated, the skin and hole stretch to a size that will accommodate insertion of the MIW.

Insert the MIW such that there is skin on top of the MIW base and suture it in place. Use tissue adhesive or cyanoacrylate to fill in the suturing holes and secure the MIW to the skin. The animal should now be left to recover for the next three to four days before the first imaging session takes place.

In preparation for the imaging session, we can describe the imaging box to make use of the imaging box. First, anesthetize the animal with isof fluorine and apply ophthalmic ointment to the eyes. Attach the isof fluorine exhaust from the anesthesia machine to the front of the imaging box.

On the outside of the inlet hole. Attach a second tube to the outlet hole of the imaging box. This tube should be attached to the gas filter and then to the vacuum.

Next, place the animal down so that the MIW is facing the bottom. Hold the box and adjust the bottom doors so that the MIW base is held and immobilized between them. The MIW base should be at the same level as the imaging box doors.

While the MIW cover slip should be just a few millimeters below this level, make sure that the positioning of the MIW between the bottom sliding doors ensures that the cover slip lies flat parallel to the bottom doors of the box. This will allow for proper focusing lower the microscope objective to allow space to put the box on top of the microscope stage. Adjust the stage so that the MIW cover slip is above the objective.

We can now focus the objective and start imaging. The imaging procedure described here is for the Leica SP five confocal microscope and should be used as a guide to optimize the experiment with a particular microscope being used. Begin by observing the tumor through the eyepiece with a green fluorescent filter.

Locate major blood vessels that are visibly flowing and laying flat in the same focal plane. Position one of the vessels in the center of the field and switch to PMT detection. We will set up sequential imaging collection for two channels.

One channel uses the 488 nanometer laser line and collects scattering from the extracellular matrix and emission from the green form of dendra two. The second channel uses the 543 nanometer laser line and collects emission from the red form of dendra.Two. Collect 3D images before photo switching.

Next, use the region of interest scan option to photo switch a chosen population of cells with the 405 nanometer laser line. Collect emission from the red form of the protein in order to monitor the switching. Now collect post switching 3D images by the same method that pre switching images were collected.

Alright, so with the use of 20%laser power in the UV to go or convert this area to photo switch more than one region in the tumor, take pictures through the oculars using a digital camera in both the green and red channels. This will help with orientation during subsequent imaging sessions. In this image, one observes a rectangular photo switch, region oriented orthogonally relative to the blood vessel, which exhibits no fluorescence.

Non-photo switch cells appear green while the scattering from the extracellular matrix is purple. The image is a maximum intensity projection of four images across the Z axis. We've just shown you how to photo switch and image RA tool labeled cells through the memory imaging window.

Using this procedure is important to compare behaviors of tumor cells in different areas of tumor over days. So that's it. Thanks for watching and good luck with your experiment.