The overall goal of this surgical procedure is to prepare the murine submandibular salivary gland for upright intravital microscopy. This method can help answer questions in any field of research that uses the submandibular salivary gland as a model organ, for instance immunology. The main advantage of this technique it allows for surgical preparation of salivary gland for upright intravital microscopy which is widely used in the field of immunology.

First, use an electric razor to remove fur from the shaving area on a C57 black six mouse. Then dab a cotton swab with hair removal cream and apply it on the shaved area. After about three minutes, use wet tissues to thoroughly remove the cream from the shaved area.

Next, remove the holders for the heating ring and the removable coverslip holder to prepare the stage for fixing the mouse. Then loosen the screws of the ear bar and the fixed coverslip holders. Next, tape a piece of gauze on the center of the stage to prepare a bedding for the mouse.

Use glue to attach a 20 millimeter diameter coverglass to the top of a cylindrical metal holder forming the lower coverglass. Then glue a 25 millimeter coverglass to the bottom of a flat metal holder forming the upper coverglass. Next, position the mouse with its back on the gauze perpendicular to the stretched string and the head centered between the ear bars.

Next, use a surgical tape to fix the upper portion of the mouse on the stage. Then hook the string in the upper front incisors, tightening the string til the jaw reaches a horizontal position. Next, use angled curved forceps to carefully pull out the tongue to ensure breathing.

Then pull the tail tightly and tape it on the stage. Next, align the ear bars such that an approximately 30 degree angle is established between the bar in the edge of the stage. To do so, draw two right and left 30 degree angles on a paper sheet.

Then position the transparent stage on the paper to align the holders. Keep moving the left and right bars inward simultaneously to fix the jaws in an immobile position and then tighten the screw. Keep an eye on the chest movement before, during, and after fixing the jaw.

Place a heating lamp nearby or a heating pad underneath the stage to heat the mouse. First, try to locate a slight bulge in the skin to identify the right submandibular salivary gland. Then use fine forceps to lift the skinfold at the center of the bulge and create an incision approximately five millimeters long.

Next, use a syringe with 25 gauge hypodermic needle filled with vacuum grease to apply a four millimeter high ring of vacuum grease along the surface of the cut. Fill the grease ring with the saline to leave the tissue moist. Then sit under a stereomicroscope and use one forceps to firmly hold the connective tissue connected to the gland and use another forceps to pull the distal connective tissue for it to rupture.

Continue disrupting the connective tissue on the top of the gland and then the medial section. Then remove the lateral connective tissue counterclockwise around the gland from the medial to the rostral section. Beginners usually struggle most with disrupting the connective tissue surrounding the gland without damaging the gland itself.

Next, use forceps to pull the connective tissue attached to the gland upwards to lift the caudal part of the gland. Then disrupt the connective tissue below the submandibular lobe from the caudal towards the rostral end. After confirming the absence of leakage from the grease ring, position the lower coverglass attached to the metal bar to its height adjustable holder.

Maintain an angle of 60 degrees with the mouse while positioning the holder from the caudal direction. Then lower the coverglass for it to contact the gland. Complete the grease ring on top of the coverglass to form a new saline reservoir.

Next, use forceps to carefully pinch the connective tissue attached to the submandibular lobe and pull it on top of the coverslip. Maintain the grease ring at a height of four millimeters in order to prevent leakage. Check such that the coverglass holder for the upper coverglass is in its upper position.

Then use a screw to attach the metal bar with the 25 millimeter upper coverglass to the holder. To position the submandibular gland in the center, use the adjustable screws of the holder to place the coverglass. Using the screws of the adjustable holder, lower the top coverglass to touch the submandibular gland.

Then note the shape and color of the gland. Next, look for possible air bubbles or drops of saline outside the reservoir to rule out leakage. Then tighten all the fixation screws.

During the routine use of this protocol, the critical step is to adjust the coverslip and the grease to form a perfect seal chamber without pressing the salivary gland too much or too little. Then insert a temperature probe inside the reservoir such that it's close to the gland and tape the probe wire to the stage. With the submandibular gland positioned in the center, apply a grease ring to the top coverslip.

Then position the heating ring on top of the coverslip and seal with the grease. Finally, tape the tubing of the heating ring to the stage. Here, the vasculature of the submandibular gland and fibroblast-like cells expressing the reporter COL1A1 green fluorescent protein is demonstrated.

In the fluorescence image, the blood vessels are labeled by intravenous injection of Texas Red Dextran conjugate whereas the fibrillar collagen present in between two lobes can be detected as the second harmonic signal. In this image, the ductal lumen is labeled with Cascade Blue Dextran after injecting it into the Wharton's duct and the green fluorescent protein tag CD8 positive T cells are also visible to show the presence of T cells in the murine salivary glands. These are the sequences of the GFP tag CD8 positive tissue resident memory T cells.

These CD8 positive T cells migrate in the submandibular salivary gland. The blood vessels in the gland are labeled by intravenous injection of Texas Red Dextran and the cell nuclei are stained with Hoechst. Once mastered, this technique can be done in 30 to 40 minutes if performed properly.

While attempting this procedure, it is important to regularly control vital signs of the mouse and the anesthesia. This procedure can be used in combination with many other methods like the adoptive transfer of fluorescently labeled immune cells to answer additional questions like the migration of immune cells in salivary gland parenchyme. This technique paved the way for researchers in the field of immunology to explore tissue resident memory T cell migration in the salivary gland.

After watching this video, you should have a good understanding of how to surgically prepare submandibular salivary gland for upright intravital microscopy.