This work was funded by Swiss National Foundation (SNF) project grant 31003A_135649, 31003A_153457 and 31003A_172994 (to JVS), and Leopoldina fellowship LPDS 2011-16 (to BS). This work benefitted from optical setups of the "Microscopy Imaging Center" (MIC) of the University of Bern.

All animal work has been approved by the Cantonal Committee for Animal Experimentation and conducted according to federal guidelines.
1. Anesthetize the Mouse
<ol>
	<li>Wear personal protective equipment, including lab coat and gloves.</li>
	<li>Mix ketamine, xylazine, and saline to a working concentration of 20 mg/mL and 1 mg/mL, respectively. Inject the working stock intraperitoneally (i.p.) at 8-10 &#181;L/g of mouse. Place the mouse back into the cage. 
	NOTE: This protocol has been...

<ol>
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J., Larsen, M., DeVine, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+matrix+and+growth+factors+in+salivary+gland+development.">Extracellular matrix and growth factors in salivary gland development.</a> Front Oral Biol. 14, 48-77 (2010).</li><li>Takeyama, A., Yoshikawa, Y., Ikeo, T., Morita, S., Hieda, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+patterns+of+CD66a+and+CD117+in+the+mouse+submandibular+gland.">Expression patterns of CD66a and CD117 in the mouse submandibular gland.</a> Acta Histochem. 117 (1), 76-82 (2015).</li><li>Hata, M., Ueki, T., Sato, A., Kojima, H., Sawa, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+podoplanin+in+the+mouse+salivary+glands.">Expression of podoplanin in the mouse salivary glands.</a> Arch Oral Biol. 53 (9), 835-841 (2008).</li><li>Proctor, G. 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V., Ariga, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+tissue+offset+correction+system+for+intravital+multiphoton+microscopy.">Real-time tissue offset correction system for intravital multiphoton microscopy.</a> J Immunol Methods. 438, 35-41 (2016).</li><li>Haghayegh Jahromi, N., Tardent, H., Enzmann, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+cervical+spinal+cord+window+preparation+allows+for+two-photon+imaging+of+T-Cell+interactions+with+the+cervical+spinal+cord+microvasculature+during+experimental+autoimmune+encephalomyelitis.">A novel cervical spinal cord window preparation allows for two-photon imaging of T-Cell interactions with the cervical spinal cord microvasculature during experimental autoimmune encephalomyelitis.</a> Front Immunol. 8, 406 (2017).</li><li>Heymann, F., Niemietz, P. 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No conflicts of interest declared.

This protocol offers a straightforward approach for in vivo imaging of murine submandibular and sublingual salivary glands using upright non-linear microscopy often used in the field of immunology. The method can be adapted for the imaging of other exocrine glands in the head and neck region. For instance, our lab has performed imaging of the lacrimal gland in an analogous manner (not shown).
Removing the connective tissue around the SMG is the most critical step of this protocol, sin...

Saliva is secreted by exocrine glands to lubricate food, protect the mucosal surfaces of the oral tract, and to deliver digestive enzymes as well as antimicrobial substances1,2. In addition to minor salivary glands interspersed in the oral submucosa, there are three bilateral sets of major glands identified as parotid, sublingual, and submandibular, according to their location1,2. Pyramid-shaped epithelial cells, organized into flask-shaped sacs (acini) or demilunes that are surrounded by myoepithelial cells and a basement membrane, secrete the serous and mucous components of saliva1. The narrow luminal space of the acini drains into intercalated ducts, which unite into striated ducts until they finally join into a single excretory duct1. The main excretory duct of the SMG is called Wharton&#39;s duct (WD) and opens into the sublingual caruncle3,4. The SMG epithelial compartment therefore represents a highly arborized structure with manifold terminal endpoints, resembling a bundle of grapes1,5,6. The SMG interstitium is composed of blood and lymphatic vessels embedded in connective tissue7 containing parasympathetic nerves8 and extracellular matrix5. Normal human and rodent salivary glands also contain T cells, macrophages, and dendritic cells9, as well as plasma cells that secrete Immunoglobulin A (IgA) into the saliva9,10. Due to its multifaceted functions in health and disease, the SMG is a subject of interest for many fields of biological research, including dentistry4, immunology11, oncology12, physiology8, and cell biology3.
Imaging of dynamic cellular processes and interactions is a powerful tool in biological research13,14. The development of deep tissue imaging and innovations inmicroscopes based on nonlinear optics (NLO), which rely on scattering or absorption of multiple photons by the sample, has allowed to directly examine cellular processes in complex tissues13,15. Absorption of multiple photons involves delivery of the total excitation energy by low energy photons, which confines fluorophore excitation to the focal plane and thus allows deeper tissue penetration with reduced photodamage and noise from out of focus excitation13,15. This principle is employed by two-photon microscopy (2PM) and allows for imaging of fluorescent specimens in depths of up to 1 mm15,16. While commercially available 2PM setups have become user-friendly and reliable, the major challenge for intravital imaging is to carefully expose and stabilize the target organ of anesthetized mice, especially for imaging of time lapse series. Several methods for digital drift correction after data acquisition have been published17,18 and we recently developed &#34;VivoFollow&#34;, an automated correction system, which counteracts slow tissue drift in real time using a computerized stage19. However, it is still critical for high quality imaging to minimize tissue motion, especially fast movements caused by breathing or heartbeat19. Preparation and stabilization procedures have been published for multiple organs, including spinal cord20, liver21, skin22, lung23, and lymph node24. Furthermore, models for rat salivary gland imaging have been developed3,25 and further refined for high resolution intravital imaging of the murine SMG tailored to an inverted microscope setup26,27,28.
Here, we present a practical and adaptable protocol for intravital imaging of the murine SMG using upright nonlinear microscopy, which is commonly used for intravital imaging in the field of immunology. To this end, we modified a widely employed immobilization stage used for popliteal lymph node preparations.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Narketan 10 %&#160; (Ketamine) 20ml (100 mg/ml)</td>
			<td>Vetoquinol</td>
			<td>3605877535982</td>
			<td></td>
		</tr>
		<tr>
			<td>Rompun 2% (Xylazine) 25 ml (20 mg/ml)</td>
			<td>Bayer</td>
			<td>680538150144</td>
			<td></td>
		</tr>
		<tr>
			<td>Saline NaCl 0.9%</td>
			<td>B. Braun</td>
			<td>3535789</td>
			<td></td>
		</tr>
		<tr>
			<td>Prequillan 1% (Acepromazine) 10 ml (10 mg/ml)</td>
			<td>Fatro</td>
			<td>6805671900029</td>
			<td></td>
		</tr>
		<tr>
			<td>Electric shaver</td>
			<td>Wahl</td>
			<td>9818L</td>
			<td>or similar</td>
		</tr>
		<tr>
			<td>Hair removal cream</td>
			<td>Veet</td>
			<td>4002448090656</td>
			<td></td>
		</tr>
		<tr>
			<td>Durapore Surgical tape (2.5 cm x 9.1 m and 1.25 cm x 9.1 m)</td>
			<td>Durapore (3M)</td>
			<td>1538-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Durapore Surgical tape (2.5 cm x 9.1 m and 1.25 cm x 9.1 m)</td>
			<td>Durapore (3M)</td>
			<td>1538-0</td>
			<td></td>
		</tr>
		<tr>
			<td>Super glue Ultra gel, instantaneous glue</td>
			<td>Pattex, Henkel</td>
			<td>4015000415040</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope cover glass slides 20 mm and 22 mm</td>
			<td>Menzel-Gl&#228;ser</td>
			<td>631-1343/ 631-1344</td>
			<td></td>
		</tr>
		<tr>
			<td>Grease for laboratories 60 g glisseal N</td>
			<td>Borer (VWR supplier)</td>
			<td>DECO514215.00-CA15</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical scissors</td>
			<td>Fine Science Tools (F.S.T )</td>
			<td>14090-09</td>
			<td>or similar</td>
		</tr>
		<tr>
			<td>Fine Forceps</td>
			<td>Fine Science Tools (F.S.T )</td>
			<td>11252-20</td>
			<td>or similar</td>
		</tr>
		<tr>
			<td>Cotton swab</td>
			<td>Migros</td>
			<td>617027988254</td>
			<td>or similar</td>
		</tr>
		<tr>
			<td>Gauze Gazin 5 x 5 cm</td>
			<td>Lohmann and Rauscher</td>
			<td>18500</td>
			<td>or similar</td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td>Leica</td>
			<td>MZ16</td>
			<td>or similar</td>
		</tr>
		<tr>
			<td>Texas Red dextran 70kDa</td>
			<td>&#160;Molecular Probes</td>
			<td>D1864</td>
			<td></td>
		</tr>
		<tr>
			<td>Cascade Blue dextran 10kDa</td>
			<td>invitrogen</td>
			<td>D1976</td>
			<td></td>
		</tr>
		<tr>
			<td>Two-photon system</td>
			<td>LaVision Biotec</td>
			<td>TrimScope I and II</td>
			<td>or similar</td>
		</tr>
		<tr>
			<td>XLUMPLANFL 20x/0.95 W objective</td>
			<td>Olympus</td>
			<td>n/a</td>
			<td>or other water immersion objective&#160;</td>
		</tr>
		<tr>
			<td>Digital thermometer</td>
			<td>Fluke</td>
			<td>95969077651</td>
			<td></td>
		</tr>
	</tbody>
</table>

preparation of murine submandibular salivary gland for upright intravital microscopy

The submandibular salivary gland (SMG) is one of the three major salivary glands, and is of interest for many different fields of biological research, including cell biology, oncology, dentistry, and immunology. The SMG is an exocrine gland comprised of secretory epithelial cells, myofibroblasts, endothelial cells, nerves, and extracellular matrix. Dynamic cellular processes in the rat and mouse SMG have previously been imaged, mostly using inverted multi-photon microscope systems. Here, we describe a straightforward protocol for the surgical preparation and stabilization of the murine SMG in anesthetized mice for in vivo imaging with upright multi-photon microscope systems. We present representative intravital image sets of endogenous and adoptively transferred fluorescent cells, including the labeling of blood vessels or salivary ducts and second harmonic generation to visualize fibrillar collagen. In sum, our protocol allows for surgical preparation of mouse salivary glands in upright microscopy systems, which are commonly used for intravital imaging in the field of immunology.

This protocol allows imaging of almost the entire dorsal or ventral side of the SMG. The field of view typically also includes the sublingual salivary gland, which differs slightly from the SMG in cellular composition4. Both glands are encapsulated by fibrillar collagen and subdivided into lobes. Most 2PM systems can produce a label-free image of fibrillar collagen by measuring the 2nd harmonic signal, but usually fluorescent molecules are needed for vis...

Watch this Scientific Journal Video about Preparation of Murine Submandibular Salivary Gland for Upright Intravital Microscopy at JoVE.com

Preparation of Murine Submandibular Salivary Gland for Upright Intravital Microscopy

We describe a protocol to surgically expose and stabilize the murine submandibular salivary gland for intravital imaging using upright intravital microscopy. This protocol is easily adaptable to other exocrine glands of the head and neck region of mice and other small rodents.

The submandibular salivary gland (SMG) is one of the three major salivary glands, and is of interest for many different fields of biological research, ...

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.

preparation-murine-submandibular-salivary-gland-for-upright

Research

JoVE Journal

Immunology and Infection

8.0K Views.  University of Bern. 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 remo...