SE is funded by Wellcome Trust grant 108906/Z/15/Z; EE is funded by UKRI/MRC grant MR/S005544/1 and by a Chancellor&#39;s Fellowship from the University of Edinburgh. Figure 1A is created with BioRender.com.

All procedures were approved by the UK Home Office and performed under PPLs PB5FC9BD2 and PP0330540. All experiments align to ARRIVE guidelines and those of the University of Edinburgh.
Wild-type mice were commercially obtained (see Table of Materials). Krt14CreER; R26mTmG mice were bred in house, by crossing Krt14CreER/+ mice31 with&#160;R26mTmG/ mTmG mice32. In all experiments, female 8-10 week old mice were used.
1. Collecting and embedding the SMG
<ol>
	<li>Euthanize the mouse/mice by exposing them to a rising concentration of carbon dioxide (CO2). Afterward, spray the incision area with 70% ethanol (EtOH) and use scissors and forceps to carefully dissect the SMG(s) from the surrounding tissue (Figure 1A). Remove any fat and connective tissue using forceps, and place the gland in a collection tube containing ice-cold Hanks Balanced Salt Solution (HBSS, see Table of Materials).</li>
	<li>Separate the gland into pieces measuring approximately 1 cm2 each using forceps.</li>
	<li>Heat 4% agarose to 50 &#176;C, and then pour it directly into a 35 mm dish.</li>
	<li>Pour a small amount of melted 50 &#176;C agarose into a separate dish and thoroughly rinse the gland by moving it around in the excess agarose. 
	NOTE: This step is necessary to remove excess liquid from the glands. An excess of liquids can prevent the glands from adhering properly to the agarose.</li>
	<li>Position 4-6 pieces of the gland into the agarose, ensuring that they lie flat and are in the same plane (Figure 1A). 
	NOTE: Ensure the glands do not touch the surface of the agar or the bottom of the plate.</li>
	<li>Place the lid on the 35 mm dish and transfer it to an icebox, covering the plate with ice.</li>
	<li>Wait for 5-10 min for the agarose to solidify.</li>
</ol>
2. Sectioning
<ol>
	<li>Use a scalpel to carefully cut around the gland-embedded agarose block, removing the block containing the tissue from the dish. Leave approximately a 5 mm border.</li>
	<li>Attach the agarose block to the stage of the vibratome (see Table of Materials) by applying a drop of superglue, ensuring that the entire bottom surface of the block is in contact with the superglue.</li>
	<li>Allow the superglue to harden for approximately 5 min. Then, fill the vibratome chamber with ice-cold 1x phosphate-buffered saline (PBS) containing 1% Penicillin-Streptomycin (P/S).</li>
	<li>Add ice to both the side and bottom of the vibratome chamber. 
	NOTE: Maintain a very cold environment and replace the ice as needed.</li>
	<li>Trim off any excess agarose and create 5 mm gaps between each piece of gland by making incisions with a scalpel. This will yield individual slices.</li>
	<li>Align the vibratome blade with the face of the agarose block and set the start and end points of the sections to achieve the desired slice thickness.</li>
	<li>Before starting the cutting process, ensure that the blade is not in contact with the block. This precaution will prevent accidentally cutting large pieces of agarose and losing parts of the sample.</li>
	<li>Cut sections with a thickness of 150 &#181;m at a low speed and high vibration (e.g., a speed of 3 and a vibration setting of 8-10 on a scale of 1-10 for each parameter) (Figure 1A).</li>
	<li>Once a section is cut and released into the surrounding PBS, collect the sections using a paintbrush and place them in a dish containing pre-warmed Roswell Park Memorial Institute (RPMI) media with P/S.</li>
</ol>
3. Culturing and staining of SMG slices
<ol>
	<li>Culturing
	<ol>
		<li>Add 1.5 mL of RPMI media to each well of a 6-well plate that contains a 0.4 &#181;m filter. Ensure there are no air bubbles trapped underneath the filter.</li>
		<li>Transfer 1-6 slices onto each filter using a paintbrush, taking care not to break the slices. 
		NOTE: Ensure that the slices are not submerged in media but are instead floating on the filter. If they are fully submerged, they may suffocate during culture.</li>
		<li>Incubate at 37 &#176;C with 5% CO2, and change the media every 3 days (Figure 1A).</li>
		<li>Irradiate experimental plates with a single dose of 10 Gy gamma radiation using a commercially available irradiator (see Table of Materials). Afterward, return them to the incubator.</li>
	</ol>
	</li>
	<li>Staining for live imaging
	<ol>
		<li>Using a paintbrush, gently lift the slices from the filter and place them into a 24-well plate containing 500 &#181;L of culture media per well, where the slices will be submerged.</li>
		<li>Add the relevant conjugated antibodies and nuclear stain (see Table of Materials) to the media at the appropriate concentrations27.</li>
		<li>Incubate the slices with antibodies for 2 h at 37 &#176;C with gentle agitation.</li>
		<li>Wash the slices by submerging them in culture media and washing for 3 rounds of 10 min washes at 37 &#176;C with gentle agitation.</li>
	</ol>
	</li>
</ol>
4. Mounting and live imaging
<ol>
	<li>Using forceps, remove the tape from one side of a double-sided imaging spacer and adhere it, adhesive side down, to the bottom of a glass-bottom 6-well plate.</li>
	<li>Pipette 50 &#181;L of media into the gap in the center of the spacer.</li>
	<li>Place the slice into the media using a paintbrush, ensuring that it lies flat without any trapped air bubbles.</li>
	<li>Using a pipette, carefully remove 20 &#181;L of media from the gap.</li>
	<li>Remove the tape from the top side of the spacer using forceps and place a 25 mm circular coverslip on top. Press around the edge of the spacer to ensure that the coverslip firmly attaches, being cautious not to break the coverslip by applying too much force (Figure 1A).</li>
	<li>Image the slice on a confocal microscope for the desired time period or intervals (e.g., every 15 min for 12 h using a 20x water objective).</li>
</ol>

Real-Time Live Imaging of Mouse Submandibular Gland Regeneration

The authors have no conflicts of interest to disclose.

The ability to culture salivary gland tissue ex vivo presents an excellent opportunity to study cell-cell interactions in the context of both homeostasis and injury response. Although intravital imaging of the mouse submandibular gland is feasible39,40, this technique depends on using fluorescent reporter mouse models to endogenously label cells of interest and must be performed under terminal anesthesia. Here, a method to culture submandibular gland slices ex vivo is described, maintaining cellular architecture and cell-cell interactions. This approach refines current live imaging techniques and provides an alternative to intravital imaging.
The long-term maintenance of tissue using this technique relies on culturing slices at an air-liquid interface. Previous explant models26,41&#160;have likely achieved successful culture for only a few days because they were submerged in media and essentially &#34;suffocated.&#34; In contrast, the use of an air-liquid interface culture system maintains tissue health and structure for an extended period, ensuring high-quality imaging. The method of mounting SMG slices before imaging, with a small amount of media and within a space-restricted chamber to keep the slice flat, is integral to the technique&#39;s success. Visualization of cells in this assay depends on endogenously labeled reporter mice or fluorescently-conjugated antibodies. The abundance of transgenic fluorescent reporter mouse models and conjugated antibodies targeting specific cell types and subsets makes this method suitable for exploring various cell-specific interactions.
While this method provides a good model of tissue in situ and ex vivo irradiation injury results in acinar and ductal structure atrophy, similar to what occurs in vivo13, some elements cannot be recapitulated ex vivo. These include the lack of functioning vasculature and neuronal input, as well as the absence of infiltrating inflammatory cells. Given the well-documented role of blood vessels and nerves in salivary gland homeostasis and regeneration26,42&#160;and the importance of migratory immune cells43, such as T and B cells, in salivary gland function, injury response, infection, and Sj&#246;gren&#39;s Syndrome (SS) pathogenesis (as reviewed in44), this assay may miss some important cellular interactions. Additionally, very fast migratory events, such as Natural Killer (NK) cell45 and Dendritic Cell (DC)46 movement, may be missed by imaging every 15 min. However, imaging intervals can be optimized to study the specific cell-cell interactions of interest, and the ability to image in 3 dimensions through z-stacks allows the assessment of 3D cell movement. Securely mounting the tissue during imaging is crucial for quantification, such as cell tracking measurements. Furthermore, although this study utilized mouse tissue, the protocol provides a viable method for studying cell-cell interactions in human salivary glands, generating valuable translational information unattainable through other methods.
While the role of tissue-resident macrophages in homeostasis and regeneration has been demonstrated in several tissues2,10,11,12, their role in the salivary glands remains largely unanswered. Although it is known that macrophages are essential for epithelial regeneration after irradiation injury13, the precise mechanisms underlying this effect remain unknown. Live imaging of salivary gland slices enables real-time visualization and analysis of complex tissue dynamics, which are often missed by traditional confocal imaging. Additionally, it is evident that macrophages undergo dynamic changes in shape while performing various functions in vivo47,48,49, and this protocol likely provides a better representation of these changes than a typical static view in fixed tissue. Future studies can utilize this technique to investigate how cell-cell communication changes across the course of homeostasis, injury, and regeneration/resolution. This approach will be useful for elucidating key signaling pathways and events that may ultimately offer therapeutic benefits.

Macrophages have been shown to play increasingly important roles in the processes of regeneration and repair, extending beyond their classical immune function1,2. Indeed, macrophages are involved in a plethora of processes related to regeneration, exhibiting critical regulatory activity in all stages of repair, as well as scar formation and fibrosis3,4. Tissue-resident macrophages are highly heterogeneous cell types with complex mechanisms driving diverse cellular phenotypes, and they play essential roles in organ development, function, and homeostasis (as reviewed in5). Tissue-resident macrophages initially arise from precursors in the yolk sac and fetal liver, and they are subsequently replaced by proliferation or by bone marrow-derived blood monocytes at varying rates, depending on the longevity of existing macrophages and the tissue or niche within which they reside6,7.
Importantly, tissue-resident macrophages are dispersed throughout all tissues and contribute to diverse organ functions. They are uniquely programmed by their microenvironment to perform niche-specific functions. For this reason, the localization of macrophages within the tissue offers insight into their function, with unique populations observed in the lung, mammary gland, intestine, skin, and muscle8,9,10. During mammary gland development, ductal macrophages are intimately associated with the ductal tree, and their depletion results in significantly reduced branching11. Furthermore, macrophages are required for morphogenesis during puberty and alveologenesis in pregnancy, where they actively monitor the epithelium. In muscle injury, a specific population of macrophages &#34;dwells&#34; within the injury site, providing a transient niche in which they supply proliferation-induced cues required for stem cell proliferation. Thus, they exhibit the specialized role of distinct macrophage populations in governing the repair process2. In the lung, a similar phenomenon occurs where interstitial macrophages prime interleukin (IL)-R1-expressing alveolar type II (AT2) cells for conversion into damage-associated transient progenitors through the release of IL-1B12. Furthermore, recent research has shown that macrophages are essential for the regeneration of the mouse submandibular salivary gland (SMG) after irradiation injury, and in their absence, epithelial regeneration is disrupted13. Taken together, this data highlights the importance of macrophage activation and function in transient inflammatory niches after tissue injury, as well as during homeostasis.
Macrophages are active cells, and their functions involve interactions with a variety of different cell types, including direct cell-cell contact14,15, as well as more indirect methods such as the secretion of soluble factors2,16, which are essential for niche regulation. While classical immunofluorescent imaging is useful to begin unraveling these interactions, it is limited by depicting only a single snapshot in time, thereby omitting numerous timepoints critical to a highly dynamic regenerative process17,18. As the importance of timing and the emergence of different waves of regeneration come into sharper focus, it is essential to dissect these processes in greater detail.
Radiotherapy is a life-saving treatment for many people diagnosed with cancer. While often effective at shrinking or eliminating the tumor(s), radiotherapy can also damage healthy tissues lying in the radiation field and elicit an immune response. Radiation injury can induce rapid macrophage recruitment, and direct and indirect immunomodulatory responses19,20. The salivary glands are often inadvertently irradiated during treatment for head and neck cancer21,22, leading to epithelial damage, cell atrophy, and fibrosis23,24, resulting in xerostomia or chronic dry mouth25.
The salivary gland is composed of a plethora of cell types and structures, including but not limited to epithelial cells (both saliva-producing acinar cells and saliva-transporting ductal cells), myoepithelial cells, epithelial progenitor cells, nerves, blood vessels, immune cells, fibroblasts, and extracellular matrix (ECM). The role and response of many of these cell types in the regenerative response have been previously described26,27,28,29,30. However, how these different cells interact during homeostasis and regeneration, and particularly how immune cells such as macrophages behave, is less well studied. This manuscript describes a newly established method to study the live interactions between SMG macrophages and other cells of interest in ex vivo tissue. The SMG is sliced on a vibratome, stained for surface markers, and imaged for up to 12 h. Using this method, phagocytosis of surrounding cells by macrophages can be observed, macrophage migration kinetics can be studied, and direct cell-cell interactions between macrophages and epithelial cells can be demonstrated.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.4 &#181;m filter cell culture inserts (Nunc)</td>
			<td>Avantor/VWR</td>
			<td>734-2240</td>
			<td>Inserts pre-packed in 6-well multidishes, 20 mm &#215; 25 mm</td>
		</tr>
		<tr>
			<td>24 well plate</td>
			<td>Corning</td>
			<td>3524</td>
			<td></td>
		</tr>
		<tr>
			<td>35 mm dish</td>
			<td>Falcon</td>
			<td>353001</td>
			<td></td>
		</tr>
		<tr>
			<td>6 well plate</td>
			<td>Corning</td>
			<td>3516</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverslips</td>
			<td>Paul Marienfeld GmbH &#38; Co. KG</td>
			<td>111650</td>
			<td>Deckglaser Cover Glasses 25 mm diameter&#160;</td>
		</tr>
		<tr>
			<td>Double-sided sticker</td>
			<td>Grace Bio-Labs</td>
			<td>654004</td>
			<td>SecureSeal Imaging Spacers SS1 x 13, 13 mm diameter x 0.12 mm depth, 25 mm x 25mm OD</td>
		</tr>
		<tr>
			<td>EtOH</td>
			<td>Scientific Laboratories Supplies</td>
			<td>CHE1924</td>
			<td>Absolute ethanol (EtOH) AR, 99.7%</td>
		</tr>
		<tr>
			<td>F4/80 antibody</td>
			<td>Invitrogen</td>
			<td>17-4801-82</td>
			<td>F4/80 Monoclonal Antibody (BM8), APC, eBioscience</td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Fine Science Tools</td>
			<td>91113-10</td>
			<td>Student Fine Forceps Straight Broad Shanks</td>
		</tr>
		<tr>
			<td>Glass bottom 6 well plate</td>
			<td>Cellvis</td>
			<td>P06-1.5H-N</td>
			<td>6 well glass bottom plate with high performance #1.5 cover glass</td>
		</tr>
		<tr>
			<td>Hanks Balanced Salt Solution (HBSS)</td>
			<td>Life Technologies</td>
			<td>14025050</td>
			<td>+calcium +magnesium, no phenol red</td>
		</tr>
		<tr>
			<td>Hoechst</td>
			<td>Sigma Aldrich</td>
			<td>14533</td>
			<td>Alternative name: bisBenzimide H 33342 trihydrochloride</td>
		</tr>
		<tr>
			<td>Ice box</td>
			<td>Fisher Scientific</td>
			<td>11339623</td>
			<td>Azlon Polyurethane Ice Buckets with Lid</td>
		</tr>
		<tr>
			<td>Imaging and analysis software</td>
			<td>Harmony</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Low Melting Agarose</td>
			<td>Merck</td>
			<td>&#160;A9414-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>Paintbrush</td>
			<td></td>
			<td></td>
			<td>Watercolour brush, 10 mm x 2mm tip</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Sigma Aldrich</td>
			<td>P4333</td>
			<td>10,000 units penicillin and 10 mg streptomycin/mL, 0.1 &#956;m filtered</td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (PBS)</td>
			<td>Life Technologies</td>
			<td>20012027</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI</td>
			<td>ThermoFisher</td>
			<td>12634010</td>
			<td>Gibco Advanced DMEM/F-12</td>
		</tr>
		<tr>
			<td>Scalpel</td>
			<td>Swann-Morton</td>
			<td></td>
			<td>Disposable scalpels, No. 11 blade</td>
		</tr>
		<tr>
			<td>Scissors&#160;</td>
			<td>Fine Science Tools</td>
			<td>14088-10</td>
			<td>Extra Narrow Scissors 10.5 cm</td>
		</tr>
		<tr>
			<td>Shepherd Mark-I-68A 137Cs irradiator</td>
			<td>JL Shepherd &#38; Associates</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Superglue</td>
			<td>Bostik</td>
			<td></td>
			<td>Multi-purpose superglue, fast setting, ultra strong</td>
		</tr>
		<tr>
			<td>Vibratome</td>
			<td>Leica</td>
			<td></td>
			<td>Leica VT 1000 S</td>
		</tr>
		<tr>
			<td>Vibratome blades</td>
			<td>Astra</td>
			<td></td>
			<td>Superior Platinum Double Edge blade</td>
		</tr>
		<tr>
			<td>Wild-type (C57BL/BJ) mice</td>
			<td>Charles River</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

interrogating cell-cell interactions in the salivary gland via ex vivo live cell imaging

Salivary gland regeneration is a complex process involving intricate interactions among various cell types. Recent studies have shed light on the pivotal role played by macrophages in driving the regenerative response. However, our understanding of this critical role has primarily relied on static views obtained from fixed tissue biopsies. To overcome this limitation and gain insights into these interactions in real time, this study outlines a comprehensive protocol for culturing salivary gland tissue ex vivo and capturing live images of cell migration.
The protocol involves several key steps: First, mouse submandibular salivary gland tissue is carefully sliced using a vibratome and then cultured at an air-liquid interface. These slices can be intentionally injured, for instance, through exposure to radiation, to induce cellular damage and trigger the regenerative response. To track specific cells of interest, they can be endogenously labeled, such as by utilizing salivary gland tissue collected from genetically modified mice where a particular protein is marked with green fluorescent protein (GFP). Alternatively, fluorescently-conjugated antibodies can be employed to stain cells expressing specific cell surface markers of interest. Once prepared, the salivary gland slices are subjected to live imaging using a high-content confocal imaging system over a duration of 12 h, with images captured at 15 min intervals. The resulting images are then compiled to create a movie, which can subsequently be analyzed to extract valuable cell behavior parameters. This innovative method provides researchers with a powerful tool to investigate and better understand macrophage interactions within the salivary gland following injury, thereby advancing our knowledge of the regenerative processes at play in this dynamic biological context.

The response of macrophages to injury in the submandibular salivary gland remains unknown. This includes whether they localize and migrate to specific structures within the gland, as well as the distance and speed at which they migrate. This has been difficult to determine through static imaging approaches.
To address this, a live imaging approach to study macrophage-epithelial cell interaction in real-time has been developed. Immunofluorescent staining of slices was combined with an endogenously labeled lineage-tracing mouse model (Figure 1A and Figure 2A). In this model, slices were exposed to 10 Gy of gamma irradiation to induce irradiation injury and were imaged at 2 h, 3 days, and 4 days post-irradiation (IR), with non-irradiated slices serving as controls. Data were acquired from four channels: Hoechst (using laser 425 to minimize phototoxicity), GFP (488), dTomato (561), and Alexa 647 (640). A z-stack was performed, and an image was captured every 2-3 &#181;m, with a total of 30 to 40 planes collected, resulting in an overall height of 80-90 &#181;m.
Using this technique and analyzing membrane-bound Tomato (mT) signal and Caspase3/7 activity, it became evident that organotypic slices retained their epithelial structure, survived in culture, and exhibited minimal cell death. The data showed that non-irradiated slices of the submandibular gland from R26mTmG mice32, cultured for 7 days, retained their mT signal and epithelial architecture (Figure 1B). However, at 3 days following ex vivo irradiation, acinar and ductal cell atrophy was evident (Figure 1B; acini and ductal structures highlighted by dashed white and green lines, respectively), consistent with in vivo radiation injury13. Apoptosis was negligible&#160;in the non-irradiated slices, but there was evidence of Caspase 3/7+ cells in the irradiated slices at 4 days post-irradiation (Figure 1C; green arrows), similar to in vivo injury33,34. Furthermore, irradiated slices recapitulated in vivo DNA damage34,35,36,37, as indicated by elevated &#947;H2AX38 compared to non-irradiated controls (Figure 1D,E). Thus, organotypic salivary gland slices exhibited good viability in culture and responded similarly to in vivo tissue to irradiation.
Following this, real-time cell-cell interactions were&#160;investigated. Macrophages directly interacting with GFP-labeled epithelial progenitor cells (Krt14CreER-GFP) were&#160;observed over a 12 h time period at 3 days post-IR (Figure 2A and Video 1). Remarkably, it was evident that macrophages remained in close proximity to epithelial progenitor cells for hours, often staying in contact for the entire 12 h imaging period. Furthermore, real-time phagocytosis of epithelial cells by macrophages was observed, confirming that macrophages carry out their traditional function in the slice culture model (Figure 2B, Videos 2 and Video 3). This technique also identified that macrophages are relatively stationary during both homeostasis and following irradiation injury, likely due to their high density within the tissue. This demonstrates, for the first time, that salivary gland macrophages do not migrate extensively during homeostasis or after irradiation injury. However, while macrophages did not show significant migration, the surrounding tissue displayed increased dynamics following irradiation, with multiple macrophages actively interacting around clusters of labeled epithelial cells. Additionally, based solely on nuclear staining, this technique allowed us to visualize cell movement within the slice over time, often with entire ducts appearing to &#39;migrate&#39; within the tissue (Video 4). Over time during the culturing process, it became evident that slices shifted from a flat to a spheroid-like morphology, suggesting that cell movement within the slice is likely due to their rearrangement into a sphere-like structure, resembling a potential reorganization event.
Finally, the live imaging data generated by this assay can be used to quantitatively measure cell behavior, such as migration. Individual cells can be detected and segmented (Figure 3A), and migration can be measured using a commercially available imaging and analysis software (see Table of Materials and Video 5). Furthermore, nearest object analysis can be undertaken to determine whether cells, in this case, macrophages, migrate closer to other cells of interest, in this case, Krt14CreER-GFP+&#160;cells, and how this dynamic changes over time (Figure 3B).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65819/65819fig1.jpg" /> 
Figure 1: Recapitulation of in vivo response in precision-cut salivary gland tissue slices. (A) Schematic of the experimental protocol. (B) Representative images of membrane-bound Tomato obtained from fresh submandibular gland (SMG) tissue, unmanipulated SMG slices cultured for 7 days, or SMG slices irradiated with a single dose of 10 Gy gamma irradiation 3 or 7 days earlier. Dashed white lines indicate example acinar structures, and dashed green lines indicate example ductal structures. Scale bar = 50 &#181;m. (C) Representative images of Caspase-3/7 expression obtained from unmanipulated SMG slices or SMG slices irradiated with a single dose of 10 Gy gamma irradiation 2 h or 4 days earlier. Green arrowheads indicate positive nuclei. Scale bar = 50 &#181;m. (D) Representative images of &#947;H2AX expression obtained from unmanipulated SMG slices or SMG slices irradiated with a single dose of 10 Gy gamma irradiation 3 days earlier. Scale bar = 20 &#181;m. (E) Representative expression of &#947;H2AX in EpCAM+ epithelial cells from unmanipulated SMG slices or SMG slices irradiated with a single dose of 10 Gy gamma irradiation 2 h and 3 days earlier. SSA = side scatter area. <a href="https://www.jove.com/files/ftp_upload/65819/65819fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65819/65819fig2.jpg" /> 
Figure 2: Live imaging of macrophage-epithelial cell interactions and phagocytosis. (A) Sequential still images of interactions between KRT14+ progenitor cells and their progeny (Krt14CreER-GFP+&#160;cells) and macrophages following irradiation. Live cell imaging captures cellular dynamics over a 90 min period. Scale bar = 20 &#181;m. Associated video is Video 1. (B) Sequential still images of a macrophage phagocytosing an epithelial cell. Live cell imaging shows the process over a 60 min period. White arrows point to the macrophage, and yellow arrows indicate the nucleus of the cell undergoing phagocytosis. Scale bar = 50 &#181;m. Associated video is Video 2. <a href="https://www.jove.com/files/ftp_upload/65819/65819fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65819/65819fig3.jpg" /> 
Figure 3: Live imaging for cellular dynamics analysis. (A) Image illustrating individual cell identification and segmentation for analyzing cellular behavior parameters, such as migration (see Video 5). Cells are pseudocolored randomly for individual cell and track distinction. Scale bar = 100 &#181;m. (B) Quantification of the distance (in &#181;m) of the nearest object to individual Krt14CreER-GFP+&#160;cells plotted over 10 time points (images captured every 5 min). Data presented as the mean value per well. <a href="https://www.jove.com/files/ftp_upload/65819/65819fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Video 1: Live imaging revealing dynamic interactions between KRT14+ progenitor cells/progeny and macrophages after irradiation. Slices were exposed to a single 10 Gy gamma irradiation dose before live imaging. Krt14CreER-GFP+&#160;cells are represented in green, macrophages (F4/80+) in red, and nuclei in cyan. The video comprises a single z-stack, spanning a 12 h culture period with images captured every 15 min.&#160;<a href="https://www.jove.com/files/ftp_upload/65819/Elder et al_Video 1.mp4" target="_blank">Please click here to download the video.</a>
Video 2: Live imaging capturing a macrophage engulfing an epithelial cell. Macrophages (F4/80+) are depicted in red, and nuclei are shown in cyan. The video consists of a single z-stack and spans a 12 h culture period with images taken every 15 min.<a href="https://www.jove.com/files/ftp_upload/65819/Elder et al_Video 2.wmv" target="_blank">Please click here to download the video.</a>
Video 3: Maximum projection live imaging showing a macrophage engulfing an epithelial cell. Macrophages (F4/80+) are displayed in red, and nuclei are shown in cyan. The video features a maximum projection of z-stack images totaling 80 &#181;m (a single plane is presented in Video 2) and spans a 12 h culture period with images taken every 15 min.<a href="https://www.jove.com/files/ftp_upload/65819/Elder at al_Video 3.wmv" target="_blank">Please click here to download the video.</a>
Video 4: Live imaging illustrating the dynamic behavior of salivary gland epithelium in culture after irradiation. Slices were subjected to a single 10 Gy gamma irradiation dose before live imaging. Macrophages (F4/80+) are represented in red, nuclei in cyan. The video comprises a single z-stack, spanning a 12 h culture period with images captured every 15 min.<a href="https://www.jove.com/files/ftp_upload/65819/Elder et al_Video 4.wmv" target="_blank">Please click here to download the video.</a>
Video 5: Quantitative cell migration tracking over time. This video demonstrates the individual tracing of cell tracks from live imaging videos. Arrows indicate cell tracks, and cells are pseudo-colored individually. The video consists of a single z-stack and spans a 1 h culture period with images taken every 15 min.<a href="https://www.jove.com/files/ftp_upload/65819/Elder at al_Video 5.wmv" target="_blank">Please click here to download&#160;the video.</a>

Watch this Scientific Journal Video about Studying Macrophage-Epithelial Cell Interactions in Salivary Gland Regeneration After Injury at JoVE.com

Author Spotlight: Studying Macrophage-Epithelial Cell Interactions in Salivary Gland Regeneration After Injury 

Immunofluorescent imaging is constrained by the ability to observe complex, time-dependent biological processes in just a single snapshot in time. This study outlines a live-imaging approach conducted on precision-cut mouse submandibular gland slices. This approach allows for the real-time observation of cell-cell interactions during homeostasis and the processes of regeneration and repair.

Interrogating Cell-Cell Interactions in the Salivary Gland via Ex Vivo Live Cell Imaging

Salivary gland regeneration is a complex process involving intricate interactions among various cell types. Recent studies have shed light on the pivotal ...

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Research

JoVE Journal

Immunology and Infection

1.3K Views.  The University of Edinburgh. Immunofluorescent imaging is constrained by the ability to observe complex, time-dependent biological processes in just a single snapshot in time. This study outlines a live-imaging approach conducted on precision-cut mouse submandibular gland slices. This approach allows for the real-time observation of cell-cell interactions during homeostasis and the processes of regeneration and repair.

Video: Author Spotlight: Studying Macrophage-Epithelial Cell Interactions in Salivary Gland Regeneration After Injury 

In vivo Imaging of Transgenic Leishmania Parasites in a Live Host

Distinct species of Leishmania, a protozoan parasite of the family Trypanosomatidae, typically cause different human disease manifestations. The most common forms of disease are visceral leishmaniasis (VL) and cutaneous leishmaniasis (CL). Mouse models of leishmaniasis are widely used, but quantification of parasite burdens during murine disease requires mice to be euthanized at various times after infection. Parasite loads are then measured either by microscopy, limiting dilution assay, or qPCR amplification of parasite DNA. The in vivo imaging system (IVIS) has an integrated software package that allows the detection of a bioluminescent signal associated with cells in living organisms. Both to minimize animal usage and to follow infection longitudinally in individuals, in vivo models for imaging Leishmania spp. causing VL or CL were established. Parasites were engineered to express luciferase, and these were introduced into mice either intradermally or intravenously. Quantitative measurements of the luciferase driving bioluminescence of the transgenic Leishmania parasites within the mouse were made using IVIS. Individual mice can be imaged multiple times during longitudinal studies, allowing us to assess the inter-animal variation in the initial experimental parasite inocula, and to assess the multiplication of parasites in mouse tissues. Parasites are detected with high sensitivity in cutaneous locations. Although it is very likely that the signal (photons/second/parasite) is lower in deeper visceral organs than the skin, but quantitative comparisons of signals in superficial versus deep sites have not been done. It is possible that parasite numbers between body sites cannot be directly compared, although parasite loads in the same tissues can be compared between mice. Examples of one visceralizing species (L. infantum chagasi) and one species causing cutaneous leishmaniasis (L. mexicana) are shown. The IVIS procedure can be used for monitoring and analyzing small animal models of a wide variety of Leishmania species causing the different forms of human leishmaniasis.

In vivo Imaging of Transgenic Leishmania Parasites in a Live Host

An in vivo imaging system is used to generate quantitative measurements of murine infection with the Trypanosomatid protozoan Leishmania. This is a non-invasive and non-lethal method for detecting parasites expressing luciferase within many tissues throughout the course of chronic Leishmania spp. infection.

Distinct species of Leishmania, a protozoan parasite of the family Trypanosomatidae, typically cause different human disease manifestations. The most ...

Use of an Optical Trap for Study of Host-Pathogen Interactions for Dynamic Live Cell Imaging

Dynamic live cell imaging allows direct visualization of real-time interactions between cells of the immune system1, 2; however, the lack of spatial and temporal control between the phagocytic cell and microbe has rendered focused observations into the initial interactions of host response to pathogens difficult. Historically, intercellular contact events such as phagocytosis3 have been imaged by mixing two cell types, and then continuously scanning the field-of-view to find serendipitous intercellular contacts at the appropriate stage of interaction. The stochastic nature of these events renders this process tedious, and it is difficult to observe early or fleeting events in cell-cell contact by this approach. This method requires finding cell pairs that are on the verge of contact, and observing them until they consummate their contact, or do not. To address these limitations, we use optical trapping as a non-invasive, non-destructive, but fast and effective method to position cells in culture.
Optical traps, or optical tweezers, are increasingly utilized in biological research to capture and physically manipulate cells and other micron-sized particles in three dimensions4. Radiation pressure was first observed and applied to optical tweezer systems in 19705, 6, and was first used to control biological specimens in 19877. Since then, optical tweezers have matured into a technology to probe a variety of biological phenomena8-13.
We describe a method14 that advances live cell imaging by integrating an optical trap with spinning disk confocal microscopy with temperature and humidity control to provide exquisite spatial and temporal control of pathogenic organisms in a physiological environment to facilitate interactions with host cells, as determined by the operator. Live, pathogenic organisms like Candida albicans and Aspergillus fumigatus, which can cause potentially lethal, invasive infections in immunocompromised individuals15, 16 (e.g. AIDS, chemotherapy, and organ transplantation patients), were optically trapped using non-destructive laser intensities and moved adjacent to macrophages, which can phagocytose the pathogen. High resolution, transmitted light and fluorescence-based movies established the ability to observe early events of phagocytosis in living cells. To demonstrate the broad applicability in immunology, primary T-cells were also trapped and manipulated to form synapses with anti-CD3 coated microspheres in vivo, and time-lapse imaging of synapse formation was also obtained. By providing a method to exert fine spatial control of live pathogens with respect to immune cells, cellular interactions can be captured by fluorescence microscopy with minimal perturbation to cells and can yield powerful insight into early responses of innate and adaptive immunity.

A method is described to individually select, manipulate, and image live pathogens using an optical trap coupled to a spinning disk microscope. The optical trap provides spatial and temporal control of organisms and places them adjacent to host cells. Fluorescence microscopy captures dynamic intercellular interactions with minimal perturbation to cells.

Dynamic live cell imaging allows direct visualization of real-time interactions between cells of the immune system1, 2; however, the lack of spatial and ...

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne relapsing fever (Borrelia spp.), Rocky Mountain Spotted fever (Rickettsia rickettsii), ehrlichiosis (Ehrlichia chaffeensis and E. equi), anaplasmosis (Anaplasma phagocytophilum), encephalitis (tick-borne encephalitis virus), babesiosis (Babesia spp.), Colorado tick fever (Coltivirus), and tularemia (Francisella tularensis) 1-8. To be properly transmitted into the host these infectious agents differentially regulate gene expression, interact with tick proteins, and migrate through the tick 3,9-13. For example, the Lyme disease agent, Borrelia burgdorferi, adapts through differential gene expression to the feast and famine stages of the tick's enzootic cycle 14,15. Furthermore, as an Ixodes tick consumes a bloodmeal Borrelia replicate and migrate from the midgut into the hemocoel, where they travel to the salivary glands and are transmitted into the host with the expelled saliva 9,16-19.
As a tick feeds the host typically responds with a strong hemostatic and innate immune response 11,13,20-22. Despite these host responses, I. scapularis can feed for several days because tick saliva contains proteins that are immunomodulatory, lytic agents, anticoagulants, and fibrinolysins to aid the tick feeding 3,11,20,21,23. The immunomodulatory activities possessed by tick saliva or salivary gland extract (SGE) facilitate transmission, proliferation, and dissemination of numerous tick-borne pathogens 3,20,24-27. To further understand how tick-borne infectious agents cause disease it is essential to dissect actively feeding ticks and collect tick saliva. This video protocol demonstrates dissection techniques for the collection of hemolymph and the removal of salivary glands from actively feeding I. scapularis nymphs after 48 and 72 hours post mouse placement. We also demonstrate saliva collection from an adult female I. scapularis tick.

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

The collection of infected tick hemolymph, salivary glands, and saliva is important to study how tick-borne pathogens cause disease. In this protocol we demonstrate how to collect hemolymph and salivary glands from feeding Ixodes scapularis nymphs. We also demonstrate saliva collection from female I. scapularis adults.

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne ...

Real-time Live Imaging of T-cell Signaling Complex Formation

Protection against infectious diseases is mediated by the immune system 1,2. T lymphocytes are the master coordinators of the immune system, regulating the activation and responses of multiple immune cells 3,4. T-cell activation is dependent on the recognition of specific antigens displayed by antigen presenting cells (APCs). The T-cell antigen receptor (TCR) is specific to each T-cell clone and determines antigen specificity 5. The binding of the TCR to the antigen induces the phosphorylation of components of the TCR complex. In order to promote T-cell activation, this signal must be transduced from the membrane to the cytoplasm and the nucleus, initiating various crucial responses such as recruitment of signaling proteins to the TCR;APC site (the immune synapse), their molecular activation, cytoskeletal rearrangement, elevation of intracellular calcium concentration, and changes in gene expression 6,7. The correct initiation and termination of activating signals is crucial for appropriate T-cell responses. The activity of signaling proteins is dependent on the formation and termination of protein-protein interactions, post translational modifications such as protein phosphorylation, formation of protein complexes, protein ubiquitylation and the recruitment of proteins to various cellular sites 8. Understanding the inner workings of the T-cell activation process is crucial for both immunological research and clinical applications.
Various assays have been developed in order to investigate protein-protein interactions; however, biochemical assays, such as the widely used co-immunoprecipitation method, do not allow protein location to be discerned, thus precluding the observation of valuable insights into the dynamics of cellular mechanisms. Additionally, these bulk assays usually combine proteins from many different cells that might be at different stages of the investigated cellular process. This can have a detrimental effect on temporal resolution. The use of real-time imaging of live cells allows both the spatial tracking of proteins and the ability to temporally distinguish between signaling events, thus shedding light on the dynamics of the process 9,10. We present a method of real-time imaging of signaling-complex formation during T-cell activation. Primary T-cells or T-cell lines, such as Jurkat, are transfected with plasmids encoding for proteins of interest fused to monomeric fluorescent proteins, preventing non-physiological oligomerization 11. Live T cells are dropped over a coverslip pre-coated with T-cell activating antibody 8,9, which binds to the CD3/TCR complex, inducing T-cell activation while overcoming the need for specific activating antigens. Activated cells are constantly imaged with the use of confocal microscopy. Imaging data are analyzed to yield quantitative results, such as the colocalization coefficient of the signaling proteins.

We describe a live-cell imaging method that provides insight into protein dynamics during the T-cell activation process. We demonstrate the combined usage of the T-cell spreading assay, confocal microscopy and imaging analysis to yield quantitative results to follow signaling complex formation throughout T-cell activation.

Protection against infectious diseases is mediated by the immune system 1,2. T lymphocytes are the master coordinators of the immune system, regulating ...

Real-time Imaging of Heterotypic Platelet-neutrophil Interactions on the Activated Endothelium During Vascular Inflammation and Thrombus Formation in Live Mice

Interaction of activated platelets and leukocytes (mainly neutrophils) on the activated endothelium mediates thrombosis and vascular inflammation.1,2 During thrombus formation at the site of arteriolar injury, platelets adherent to the activated endothelium and subendothelial matrix proteins support neutrophil rolling and adhesion.3 Conversely, under venular inflammatory conditions, neutrophils adherent to the activated endothelium can support adhesion and accumulation of circulating platelets. Heterotypic platelet-neutrophil aggregation requires sequential processes by the specific receptor-counter receptor interactions between cells.4 It is known that activated endothelial cells release adhesion molecules such as von Willebrand factor, thereby initiating platelet adhesion and accumulation under high shear conditions.5 Also, activated endothelial cells support neutrophil rolling and adhesion by expressing selectins and intercellular adhesion molecule-1 (ICAM-1), respectively, under low shear conditions.4 Platelet P-selectin interacts with neutrophils through P-selectin glycoprotein ligand-1 (PSGL-1), thereby inducing activation of neutrophil &beta;2 integrins and firm adhesion between two cell types. Despite the advances in in vitro experiments in which heterotypic platelet-neutrophil interactions are determined in whole blood or isolated cells,6,7 those studies cannot manipulate oxidant stress conditions during vascular disease. In this report, using fluorescently-labeled, specific antibodies against a mouse platelet and neutrophil marker, we describe a detailed intravital microscopic protocol to monitor heterotypic interactions of platelets and neutrophils on the activated endothelium during TNF-&alpha;-induced inflammation or following laser-induced injury in cremaster muscle microvessels of live mice.

Here we report an experimental technique of fluorescence intravital microscopy to visualize heterotypic platelet-neutrophil interactions on the activated endothelium during vascular inflammation and thrombus formation in live mice. This microscopic technology will be valuable to study the molecular mechanism of vascular disease and to test pharmacologic agents under pathophysiological conditions.

Interaction of activated platelets and leukocytes (mainly neutrophils) on the activated endothelium mediates thrombosis and vascular inflammation.1,2 ...

In vivo Imaging Method to Distinguish Acute and Chronic Inflammation

Inflammation is a fundamental aspect of many human diseases. In this video report, we demonstrate non-invasive bioluminescence imaging techniques that distinguish acute and chronic inflammation in mouse models. With tissue damage or pathogen invasion, neutrophils are the first line of defense, playing a major role in mediating the acute inflammatory response. As the inflammatory reaction progresses, circulating monocytes gradually migrate into the site of injury and differentiate into mature macrophages, which mediate chronic inflammation and promote tissue repair by removing tissue debris and producing anti-inflammatory cytokines. Intraperitoneal injection of luminol (5-amino-2,3-dihydro-1,4-phthalazinedione, sodium salt) enables detection of acute inflammation largely mediated by tissue-infiltrating neutrophils. Luminol specifically reacts with the superoxide generated within the phagosomes of neutrophils since bioluminescence results from a myeloperoxidase (MPO) mediated reaction. Lucigenin (bis-N-methylacridinium nitrate) also reacts with superoxide in order to generate bioluminescence. However, lucigenin bioluminescence is independent of MPO and it solely relies on phagocyte NADPH oxidase (Phox) in macrophages during chronic inflammation. Together, luminol and lucigenin allow non-invasive visualization and longitudinal assessment of different phagocyte populations across both acute and chronic inflammatory phases. Given the important role of inflammation in a variety of human diseases, we believe this non-invasive imaging method can help investigate the differential roles of neutrophils and macrophages in a variety of pathological conditions.

In vivo Imaging Method to Distinguish Acute and Chronic Inflammation

We describe a non-invasive imaging method for distinguishing inflammatory stages. Systemic delivery of luminol reveals areas of acute inflammation dependent upon MPO activity in neutrophils. In contrast, injection of lucigenin allows for visualization of chronic inflammation dependent upon Phox activity in macrophages.

Inflammation is a fundamental aspect of many human diseases. In this video report, we demonstrate non-invasive bioluminescence imaging techniques that ...

Murine Lymphocyte Labeling by 64Cu-Antibody Receptor Targeting for In Vivo Cell Trafficking by PET/CT

This protocol illustrates the production of 64Cu and the chelator conjugation/radiolabeling of a monoclonal antibody (mAb) followed by murine lymphocyte cell culture and 64Cu-antibody receptor targeting of the cells. In vitro evaluation of the radiolabel and non-invasive in vivo cell tracking in an animal model of an airway delayed-type hypersensitivity reaction (DTHR) by PET/CT are described.
In detail, the conjugation of a mAb with the chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) is shown. Following the production of radioactive 64Cu, radiolabeling of the DOTA-conjugated mAb is described. Next, the expansion of chicken ovalbumin (cOVA)-specific CD4+ interferon (IFN)-&#947;-producing T helper cells (cOVA-TH1) and the subsequent radiolabeling of the cOVA-TH1 cells are depicted. Various in vitro techniques are presented to evaluate the effects of 64Cu-radiolabeling on the cells, such as the determination of cell viability by trypan blue exclusion, the staining for apoptosis with Annexin V for flow cytometry, and the assessment of functionality by IFN-&#947; enzyme-linked immunosorbent assay (ELISA). Furthermore, the determination of the radioactive uptake into the cells and the labeling stability are described in detail. This protocol further describes how to perform cell tracking studies in an animal model for an airway DTHR and, therefore, the induction of cOVA-induced acute airway DHTR in BALB/c mice is included. Finally, a robust PET/CT workflow including image acquisition, reconstruction, and analysis is presented.
The 64Cu-antibody receptor targeting approach with subsequent receptor internalization provides high specificity and stability, reduced cellular toxicity, and low efflux rates compared to common PET-tracers for cell labeling, e.g.64Cu-pyruvaldehyde bis(N4-methylthiosemicarbazone) (64Cu-PTSM). Finally, our approach enables non-invasive in vivo cell tracking by PET/CT with an optimal signal-to-background ratio for 48 h. This experimental approach can be transferred to different animal models and cell types with membrane-bound receptors that are internalized.

Murine Lymphocyte Labeling by 64Cu-Antibody Receptor Targeting for In Vivo  Cell Trafficking by PET/CT

Following the preparation of a 64Cu-modified monoclonal antibody binding to a transgenic murine T cell receptor, T cells are radiolabeled in vivo, analyzed for viability, functionality, labeling stability and apoptosis, and adoptively transferred into mice with an airway delayed-type hypersensitivity reaction for non-invasive imaging by positron emission tomography/computed tomography (PET/CT).

This protocol illustrates the production of 64Cu and the chelator conjugation/radiolabeling of a monoclonal antibody (mAb) followed by murine lymphocyte ...

Ex Vivo Infection of Human Lymphoid Tissue and Female Genital Mucosa with Human Immunodeficiency Virus 1 and Histoculture

Histocultures allow studying intercellular interactions within human tissues, and they can be employed to model host-pathogen interactions under controlled laboratory conditions. Ex vivo infection of human tissues with human immunodeficiency virus (HIV), among other viruses, has been successfully used to investigate early disease pathogenesis, as well as a platform to test the efficacy and toxicity of antiviral drugs. In the present protocol, we explain how to process and infect with HIV-1 tissue explants from human tonsils and cervical mucosae, and maintain them in culture on top of gelatin sponges at the liquid-air interface for about two weeks. This non-polarized culture setting maximizes access to nutrients in culture medium and oxygen, although progressive loss of tissue integrity and functional architectures remains its main limitation. This method allows monitoring HIV-1 replication and pathogenesis using several techniques, including immunoassays, qPCR, and flow cytometry. Of importance, the physiologic variability between tissue donors, as well as between explants from different areas of the same specimen, may significantly affect experimental results. To ensure result reproducibility, it is critical to use an adequate number of explants, technical replicates, and donor-matched control conditions to normalize the results of the experimental treatments when compiling data from multiple experiments (i.e., conducted using tissue from different donors) for statistical analysis.

Ex Vivo Infection of Human Lymphoid Tissue and Female Genital Mucosa with Human Immunodeficiency Virus 1 and Histoculture

Infection of human tissues with human immunodeficiency virus (HIV) ex vivo provides a valuable 3D model of virus pathogenesis. Here, we describe a protocol to process and infect tissue specimens from human tonsils and female genital mucosae with HIV-1 and maintain them in culture at the liquid-air interface.

Histocultures allow studying intercellular interactions within human tissues, and they can be employed to model host-pathogen interactions under ...

A Method for the Measurement of Salivary Gland Function in Mice

Patients with Sj&#246;gren&#39;s syndrome, an autoimmune disease affecting the exocrine glands, develop salivary gland inflammation and have reduced saliva production. Similarly, saliva production is severely compromised in patients receiving radiation treatment for head and neck cancers. Rodent models, developed to mimic these clinical conditions, facilitate an understanding of the disease pathogenesis and allow for the development of new therapeutic strategies. Therefore, the ability to accurately, reproducibly, and repeatedly measure salivary gland function in animal models is critical. Building on procedures previously described in the literature, a method was developed that meets these criteria and was used to evaluate salivary gland function in mice. An additional advantage of this new method is that it is easily mastered, and has little inter-operator variation. Salivary gland function is evaluated as the amount (weight or volume) or rate (mL/min) of saliva produced in response to pilocarpine stimulation. The collected saliva is a good source for the analyses of protein content, immunoglobulin concentrations, and other biomolecules.

Salivary gland hypofunction is a frequent consequence of autoimmune disease and radiation therapy. Reproducible evaluation of salivary gland function in mouse models of these diseases is a technical challenge. Here, a simple method for accurate and reproducible measurement of saliva production in mice is described.

Patients with Sj&#246;gren&#39;s syndrome, an autoimmune disease affecting the exocrine glands, develop salivary gland inflammation and have reduced ...

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.

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, ...