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First, the bottom of the dish is broken away from the walls and cut into rectangles of about 15 x 10 mm. Then, the edges are rounded and polished by using fine sandpaper. Four spacers 1. By using fine sandpaper, the surface of the spacers and the four corners of the plastic piece are polished. The spacers are then glued by using gelatinous superglue, and later smoothed using fine sandpaper on a flat surface.

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Note, that for older animals, the size of the holders and the spacers may have to be increased to accommodate slightly larger glands. Preparation of the optical coupling gel.

Then, 0. The beaker should be covered and placed on a stirring hot plate lowest heat setting for several hours or until all the carbomer clumps are completely dissolved.

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Lastly, Trietanolamine is added drop-wise while gently stirring, until a pH of about 7. Part 2: Animals and Anesthesia Before conducting any experiments with animals, the protocols should be approved by your local animal care and ethics committee. Importantly, researchers should be properly trained to perform surgical procedures and should consult with their local animal care committee or institution veterinarian before proceeding with the procedure.

Note: it usually take min for the animal to become fully anesthetized and prepped for imaging Mice are housed in a controlled environment in the animal facility and are allowed to acclimate for one week prior to the experiment.

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This step is necessary for imaging the SGs since any distress results in enhanced secretory activity Water and food are provided ad libitum. Males from 2 -5 months old are used for imaging experiments g.

Prior to anesthesia, weigh the animals to determine the proper dose of anesthetic. Since the stress induced by administration of injectable anesthetics may impair the secretory activity of the SGs, induce pre-anesthesia by isoflurane inhalation, followed by the injection of anesthetics.

To this aim, place the mice very gently into the vaporizer chamber, and turn on the oxygen flow mmHg. This mixture is further diluted with saline and an appropriate amount is injected based on the animal weight.

This dilution helps in preventing accidental overdose. Note that the mixture loses its effectiveness after 20 min. Check the depth of the anesthesia every 15 min by pinching the paw and observing the response. Note: Always administer ophthalmic ointment to prevent eye dryness. Anesthetics induce hypothermia, which can affect the reproducibility of the experiments and the health of the animal. Therefore, mice should be kept under the heat lamp or on a heated pad for the entire duration of the experiment.

Note: the heat lamp should be at least inches from the animal to prevent burning The body temperature of the animals has to be monitored with a thermometer equipped with a rectal temperature probe. Part 3: Animal Surgery and Positioning for Intravital Microscopy Prepare the workspace with all the surgical instruments Table 1 that should be clean and sterile. Shave the neck area of the mouse with an electric clipper. Dry the excess with a Kimwipe.

With dull curved tweezers lift up a small piece of skin at the midline approximately at the lower end of the masseter muscles , and make a small incision Figure 1B. Insert the scissors into the opening and separate the skin away from the underlying tissue by opening the scissor blades.

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Avoid damaging the underlying glandular tissue by pointing the scissors up towards the underside of the skin. Repeat this step several times until the skin is separated from the deeper tissues Figure 1B. Using scissors, cut out a strip of loosened skin about 3 mm wide and 10 mm long.

The skin should be cut so that the opening starts near the base of the SGs where the nerve, the duct and the blood vessels connect the gland to the rest of the body. After cleaning the wound area, the resultant opening will be oval in shape and measure roughly 8 x 12 mm. Both SGs will be visible after the opening Figure 1B. With a syringe apply the optical coupling gel into the middle of the cut and wipe it towards the outside with sterile gauze.

Clean pieces of gauze should be used for each wipe to prevent introduction of hair or blood into the exposed tissue. Repeat this step until all blood and loose hair is removed. Using 7 tweezers, separate the connective tissue away from right submandibular gland all the way to the base of the gland alternatively this protocol can be used on the left SGs.

First, find the tip of the SG and grab the connective tissue a few millimeters above the tip of the gland. Tear the connective tissue around the gland without injuring the parenchyma. Figure 1B. Once the gland is exposed, gently separate the connective tissue from the gland.

If bleeding occurs, wash the blood away with saline. Make sure that all the connective tissues are separated and the gland is fully exposed. Keep the gland moist by applying optical coupling gel regularly. The temperature of the chemical heat pack can be adjusted by the size of the cuts that expose the inner pouch to the atmosphere.

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Gauze provides some insulation of the heat pack from the metal stage. Cover the top of the heat pack with a thin piece of gauze as well. Place the animal on its side right side for the right gland and onto the gauze.

Position the animal with the submandibular salivary gland placed in the middle of the coverslip Figure 1C. The animal should be secured and stabilized in this position before immobilizing the gland. Place a piece of masking tape on the ventral side of the front right paw of the mouse. Hook the incisors of the mouse by a silk suture Figure 1C, inset.

Create some tension between the silk thread and the paw and attach them onto the stage by masking tape. The SG should be resting naturally in the middle of the coverslip. The head and the thorax should be additionally stabilized by plastic wedges that can be coupled to the stage. Place a small piece of lens cleaning tissue over the gland Figure 1C. Extend the gland slightly and place a customized holder over the gland. Tightly couple the stabilizer to the stage with masking tape.

Coupling can be improved by a metal clamp connected to the stage Figure 1C. Tension and sharp angles should be avoided as they may compromise the blood flow. Immediately after the stabilization inspect the blood flow either by examining the gland visually it should retain pink coloration or by epifluorescence. Cover the animal with gauze and a heated pad. Measure the temperature of the exposed gland by using a thermometer equipped with a small flexible thermocouple probe placed in contact with one side of the exposed gland.

Find the surface of the SG, and assess the blood flow in the capillaries and the overall morphology of the tissue.