The overall goal of this procedure is to expose and stabilize the popal lymph node for intra vital imaging. This is accomplished by first creating a holder for the mouse, which allows for the stabilization of the leg during the procedure. The second step is to properly place the anesthetized mouse onto the holder.
The third step is to carefully expose the popal lymph node without damaging it or any of the surrounding vasculature. The final step is to perform intravital imaging on the exposed pubal lymph node. Ultimately, the real-time migration and interactions of immune cells in the lymph node can be observed and analyzed through intravital microscopy.
Visual demonstration of this method is critical. As the surgical steps are difficult to learn and providing adequate stabilization of the lymph node without damaging it is crucial. Demonstrating the procedure will be Rachel Liu.
A technician in our lab Begin by placing the cover of a 100 millimeter glass Petri dish right side up on top of an upside down 100 millimeter plastic Petri dish lid with the glass barely touching the center point of the plastic dish. Using the glass as a stencil trace a crescent line across the plastic dish. Next, use a hand drill to cut the 100 millimeter plastic dish lid into a crescent shaped platform.
Drill two holes into the platform and then secure the plastic lid to the edge of the glass Petri dish using a strong adhesive. Finally, cut two domed cap PCR caps off of their tubes at the hinges and glue the caps one centimeter apart in the center of the bottom dish. Once the mouse has been anesthetized, secure the gas mask over the animal's nose with tape.
Adjust isof fluorine levels accordingly to ensure the animal is fully sedated. Next, use an electric trior to remove the hair on the right hind leg and inguinal area of the mouse. Brush the loose hair away and then use a cotton swab to gently apply a modest coat of hair removal cream onto the shaven area.
One minute After the initial application, remove the detory cream and clean the exposed skin with a damp paper towel. When the mouse is dry, make a small two to three millimeter incision with scissors at the right knee to expose the extensor tendon. Now, apply vet bond along the center of the mouse holder with the mouse body and leg will be positioned carefully.
Secure the mouse onto the holder, keeping the right knee down for exposure of the right popal fossa. Then secure the right knee tendon with vebo in between the two skin flap holders to help stabilize the imaging field. Next, stretch and tape the arms and left leg of the mouse to the upper platform of the holder.
Insert two metal twist ties through the holes and use the ties to secure the gas mask to the lid. Finally, place the holder under the dissection microscope. Place the tail in a position that will contribute to the greatest stability of the right leg, typically above the head as shown here.
And tape the tail down. While under the dissection scope, maintain the mouse body temperature using a space heater. Begin the procedure by sterilizing the skin with Betadine using sterile scissors.
Make a midline incision through the skin at the right midcalf and continue cutting vertically up to the superior portion of the right thigh. Then make two horizontal skin incisions at the top of the vertical incision line to create skin flaps on either side. Preserve tissue moisture by constantly applying warm PBS to the exposed tissue.
Pull the skin taut, then apply vet bond to the flaps to further promote leg stability. Continue to glue down other areas of the skin to increase the stability of the leg before exposing the lymph node. Exposing the lymph node without damaging the surrounding tissues is the trickiest part of this procedure.
Use extra caution when separating the adipose tissue and muscles being careful not to damage the surrounding blood vessels and lymph vessels. The best way to do this is to lift a couple of muscle strands at a time up and away from the lymph node when separating them. Now, use micro dissecting tweezers and forceps to carefully splay the surrounding adipose tissues and muscles exposing the popal lymph node without jeopardizing the integrity of the afferent and ENT blood vessels and the afferent lymphatic vessels.
Once the lymph node is adequately exposed and stability is achieved, promptly transfer the entire mouse holder assembly onto the two photon imaging microscope stage at enough sterile 37 degrees Celsius PBS to the environmental microscope chamber to submerge the lymph node. Maintain the PBS temperature at 37 degrees Celsius using a heating pad with a feedback probe. Place a separate temperature probe in the mouse holder to confirm the temperature of the PBS remains in the range of 36.5 to 37.5 degrees Celsius.
A rectal probe also may be used to monitor the core body temperature of the experimental animal. Use an epi fluorescent lamp to help guide the lymph node placement under the objective. Then acquire fluorescent image stacks using time intervals appropriate for the desired cellular interaction.Action.
Monitor the status of the animal in the microscope chamber frequently by visual inspection. This 3D snapshot was taken from a two photon laser scanning microscopy imaging sequence of the popal lymph node in a mouse adoptively transferred with one times 10 to the seventh GFP positive lymphocytes one day prior to imaging. Even without other landmarks.
Structures such as the B-cell follicles denoted here by the dotted lines can be discerned easily by the round spherical cell. Accumulation in this figure tracks of lymphocyte migration during one hour of continuous imaging are shown. This allows for tracking of cell migration patterns as well as complex analysis of cell behavior such as speed, directionality, and cell to cell interactions.
Note the OID shape of the migrating lymphocytes. Here, a distribution of the overall lymphocyte migration speed is presented of adoptively transferred lymphocyte migration tracked in a sample pubity a lymph node of a total of 15, 125 tracks analyzed. The mean speed was determined to be between six to 14 micrometers per minute.
This graph shows the differential cellular migration speed distribution of cells found in the B cell follicle represented by the open data bars and the T-cell zone represented by the closed data bars. On average, the frequency of cellular migration in the B-cell follicle exceeded that of the cellular migration tracked in the T-cell zone. Although the cells in the T-cell zone migrated at higher average speeds in this time, lapse intravital two photon laser scanning microscopy movie of a mouse popal lymph node, a total of one times 10 of the seventh lymphocytes were isolated from a ubiquitin GFP positive donor mouse and adoptively transferred intravenously into the recipient mouse 24 hours before imaging as seen in the movie.
This technique makes it possible to dynamically visualize lymphocyte behavior in the lymph node. Here, a zoomed in view of the imaging sequence of the B-cell follicle T-cell zone border area from the previous video is shown. Through careful analysis, one can differentiate cell behaviors in the different microenvironments of the lymph node.
After watching this video, you should have a good understanding of how to create a mouse holder suitable for imaging the popliteal lymph node, how to surgically expose the popliteal lymph node and how to perform intra vital microscopy in order to observe the behavior of immune cells in C two.
