The overall goal of this procedure is to perform intra vital immunofluorescence imaging of the ear of melanoma bearing mice to study the dynamics of matrix components of tumor tissue. This is accomplished by first inoculating a mouse ear with GFP expressing B 16 F 10 melanoma cells. Next surgery is performed on the mouse ear in order to separate the ventral from the dorsal ear skin and expose the tumor tissue.
Then immuno staining is performed on the exposed tissue of the dorsal ear. Finally, the animal is prepared for intra vital imaging. Ultimately, fluorescent stereo microscopy or multi photon microscopy is used to show localization and dynamic behavior of tumor cells and their matrix components.
The main advantage of this technique compared to the other live imaging methods is that we can image the dynamic interactions of tumor cells with the stromal associated cells such as immune cells, but it's in the context of fibrillar and mesh like extracellular matrix. So this method can help you address key questions in the field of tumor biology, such as how tumor cells interact with their physical microenvironment, which in turn may provide important insights into how tumors progress. Some part of the procedure will be demonstrated by Vita Kki, a senior scientist of our laboratory To prepare tumor cells for injection begin by culturing B 16 F 10 GFP melanoma cells.
After centrifuging the cells transfer them to a 1.5 milliliter einor tube and spin again, remove all supernatant except a thin layer of medium that stays just above the pellet, resuspend the cells into a thick cell slurry next to load the cell slurry into a 10 microliter volume. Hamilton syringe. Remove the tip cap from an attached 33 gauge needle and load 20 microliters of the slurry on top of the tip chamber.
Then pull back on the plunger until five microliters of slurry is loaded inside the syringe. After anesthetizing a C 57 black six mouse with injectable anesthesia, shave the mouse head, dete the hairs on the head and around the ear, and rinse with water. Using adhesive tape, fix the proximal edge of the ear on the tip of an index finger.
Insert the Hamilton syringe needle between the dorsal dermis and the cartilage of the ear of a C 57 black six mouse penetrating the ear proximal to distal for approximately two to three millimeters. Inject the cell slurry and slowly retract the needle from the skin. Let the tumor cells form a solid tumor over the course of seven to nine days, using a fluorescence microscope to follow the tumor growth.
After using a mixture of humidified oxygen and iso fluorine to anesthetize the mouse, place the mouse on a 37 degrees Celsius heating pad and perform a gentle toe pinch. To confirm that the animal is sufficiently anesthetized. Apply ophthalmic ointment to the eyes and keep the mouse on a rectal thermistor controlled heating pad throughout the entire procedure.
Next, gently place the tumor bearing ear on a stack of glass slides. Then use small strips of adhesive tape to fixate the anterior and posterior edges of the ear to the stack using a scalpel, cut the ventral skin of the ear along the anti helix of the mouse pinna. Remove the tapes from the ear with the help of curved tweezers.
Gently peel off the ventral dermis and the cartilage from the dorsal dermis. Use ringer's buffer to wash the ear to carry out immunofluorescent staining. Apply primary antibodies targeting extracellular matrix molecules in blocking buffer to the exposed ear, and place a cover slip on top.
Incubate for 15 minutes before using approximately five milliliters of ringers buffer to wash the ear twice. Apply appropriate secondary antibodies or strep Aden in blocking buffer. Apply a cover slip and incubate for 15 minutes before washing twice As before, fold the open dermal area into the EM mencia conki and use sterile wipes to dry the outer unopened ear dermis.
Place the ear on a stack of glass slides and apply 0.5 microliters of surgical glue to the anterior and posterior dorsal edges to immobilize it for short term imaging of two hours or less. Add freshly prepared sterile as sorbate ringers buffer on top of the immobilized ear and apply a cover slip. Then use a fluorescent stereo microscope with a two lens to image the ear.
For long-term imaging. Place the outlet of a needle attached to a reservoir containing a sorbate ringers under the cover slip approximately 0.5 centimeters away from the ear. Use a peristaltic pump at a speed of one microliter per minute to constantly deliver the buffer to the chamber.
Open the acquisition software. Adjust the gain fluorescence intensity, magnification and exposure time image, several fields that contain tumor cells, as well as stained extracellular matrix proteins to carry out intra vital imaging using multi photon microscopy with silicon grease. Starting at the base of the ear.
Build a circular wall of about two centimeters diameter and two to three millimeters height around the ear. Fill the circle with a sorbate ringers. Place the mouse on the stage on a heating pad, set at 37 degrees Celsius and configure the settings of the microscope according to the text protocol.
As shown in this figure, many structures can be distinguished based on their morphology after immuno staining. For basement membrane components such as collagen four or prolean, most importantly, structural proteins that normally cannot be detected by SHG. The classic matrix detection method can be visualized.
For example, we found that tens and C is deposited in different locations of tumor stroma from fibrillar collagens forces that develop within the tumor microenvironment may lead to expansion or contraction of the tumor matrix, and as a consequence, remodeling of the tumor vasculature similar to wound healing. Here we show that we can perform two photon time-lapse microscopy, while simultaneously imaging immuno labeled tenas and C matrix with minimal photo bleaching of Fluor fours, even in high photon density to photon microscopy. In this video, tumor cells that were overlaid on the exposed dermis prestained for collagen four adhered to the tissue and in groups started to migrate collectively along the basement membrane of blood vessels and adipose sites.
Once mastered the entire procedure of opening the ear and immuno staining takes two hours. After watching this video, you should have a good understanding of how to perform intravital immuno staining to study cell matrix interactions in tumors using epi fluorescence or two photon microscopy.
