This work is supported by grants from NCI 1R01CA154656 , NIAID 1R21AI092299, Cancer Research Institute, St. Baldrick's Foundation, Dana Foundation, Gabrielle's Angel Foundation, and Hyundai Motors of America "Hope-on-Wheels" Program.

1. Mouse Holder Assembly
<ol>
	<li>Overlay the cover of a 100-mm glass Petri dish on top of a 100-mm plastic Petri dish lid facing down. The glass should be barely touching the center point of the plastic dish. Using the glass as a stencil, trace a mark onto the plastic dish.</li>
	<li>Use a hand drill (i.e. Dremel) to remove a portion of a 100-mm plastic dish lid to create a crescent-shaped platform. This crescent-shaped plastic lid will serve as a support for the upper torso of the mouse (Figure 1a). Drill two holes on the crescent-shaped lid to secure the gas mask with a metal twist tie. Secure the plastic lid to the edge of the glass Petri dish using super glue or an equivalently strong adhesive (Figure 1b).</li>
	<li>Glue the lids of two domed-cap PCR tubes (cut at the hinge) 1 cm apart to the center of the bottom dish for the purpose of anchoring popliteal skin flaps.</li>
</ol>
2. Mouse Preparation
Note: With sufficient practice, one should be able to perform the mouse preparation and surgical steps in 20-30 minutes.
<ol>
	<li>Anesthetize the mouse with isoflurane (2 to 2.5% for induction, 1.5 to 2% for surgery / imaging) admixed in 1:1 O2 : air mixture at a flow rate of 1L/min using an IACUC- approved procedure. Once the mouse is anesthetized, secure a gas mask over the nose with tape. Determine if the mouse is fully anesthetized by the lack of response to toe and/or tail pinches. The level of isoflurane may be adjusted accordingly to ensure the animal is fully sedated, with a steady, non-labored respiratory rate between 60-80 breaths/min.</li>
	<li>Use an electric trimmer to remove hair on the right hind leg and inguinal area of the mouse.</li>
	<li>Brush away loose hair and gently apply a modest coat of Nair lotion onto the shaven area with a cotton swab. After one minute of initial application, remove the Nair and clean the exposed skin with a damp paper towel. Make sure the mouse is clean and dry before proceeding.</li>
	<li>Make a small 2 to 3 mm incision with scissors at the right knee to expose the extensor tendon.</li>
	<li>Apply Vetbond along the center of the mouse holder where the mouse body and leg will be positioned. Carefully secure the mouse onto the holder, with the right knee down, to expose the right popliteal fossa. Secure the right knee tendon with Vetbond in between the 2 skin flap holders to help stabilize the imaging field.</li>
	<li>Stretch and tape the arms and left leg to the upper platform of the holder.</li>
	<li>Use a twist tie to secure the gas mask in place.</li>
	<li>Place the holder under the dissection microscope. The LN must remain absolutely still, independent of the breathing motion of the mouse. Therefore, precautions must be taken while performing the surgery to optimize the stability of the leg. Determine the position of the tail (typically above the head) that will contribute to the greatest stability to the right leg and tape the tail down.</li>
</ol>
3. Surgery
<ol>
	<li>While under the dissection scope, maintain mouse body temperature using a space heater or a heating pad. For successful imaging of the popliteal LN, it is critical to sustain proper body temperature throughout the surgery as well as to preserve tissue moisture by constantly applying warm PBS to the exposed tissue.</li>
	<li>Sterilize the skin with Betadine. Using sterile scissors, make a midline incision through the skin at the right mid-calf, and continue cutting vertically up to the superior portion of the right thigh.</li>
	<li>Make two horizontal skin incisions at the top of the vertical incision line to create skin flaps on either side.</li>
	<li>Retract and glue down both skin flaps with Vetbond. Pulling the skin taut prior to applying Vetbond will further promote leg stability; however, make sure that the skin tension does not occlude blood flow (i.e. changes in the vessel color or diameter). Continue to glue down other areas of the skin to ensure stability of the leg before exposing the LN. The size of the mouse will determine how much extra skin is needed to glue down. Typically, bigger mice will require more skin to be glued to the holder.</li>
	<li>The LN should lie within the popliteal fossa either to the right or left of the popliteal vein, depending on the placement of the mouse on the holder. Carefully separate the LN from surrounding adipose tissues and muscles using micro-dissecting tweezers and forceps. To minimize bleeding and trauma, use the splaying techniques with micro-dissecting tweezers to separate tissues. Carefully expose the popliteal LN without jeopardizing the integrity of the afferent and efferent blood vessels and the afferent lymphatic vessels.</li>
	<li>For added tissue stability, a square cover glass may be positioned over the moist LN, just barely touching the LN while avoiding vessel occlusion. The cover glass may be secured with modeling clay on either side of the mouse.</li>
	<li>Fluorescent vessel dyes of various sizes (e.g. TRITC-dextran, minimum of 70kDa) may be introduced intravenously at this point to help highlight the structural relationship and integrity of the LN during imaging.</li>
</ol>
4. 2-Photon Imaging Acquisition**
<ol>
	<li>Once the LN is adequately exposed and the stability is achieved, promptly transfer the entire mouse holder assembly onto the microscope stage fitted with a programmable temperature feedback controlled heating pad in an environmental microscope chamber kept at 37&deg;C. Add enough sterile warm (37&deg;C) PBS or HBSS to submerge the LN. The volume will change depending on the size and placement of the mouse. Alternatively, warm PBS or HBSS may be applied directly to the dissected LN through a fine glass pipette fitted to a peristaltic pump and taped to the column of the immersion lens objective. The flow of the liquid should be such that a stable water column can be maintained between the lens and the tissue.</li>
	<li>Maintain the PBS or HBSS temperature at 37&deg;C using a heating pad with a feedback probe. A separate temperature probe should be placed in the mouse holder to confirm the temperature of the PBS in the range of 36.5 to 37.5&deg;C. A rectal probe may also be used to monitor core body temperature of the experimental mouse throughout the imaging session.</li>
	<li>Use an epi-fluorescent lamp to help guide LN placement under the objective. Acquire fluorescent image stacks using time intervals that are appropriate for the desired cellular interaction (typically 10 seconds to 1 minute in between each xyz image stack).</li>
	<li>Monitor the status of the animal in the microscope chamber frequently by visual inspection or by using an animal monitoring system. Determine if the mouse is fully anesthetized by the lack of response to toe and/or nail pinches, and a steady, non-labored respiratory rate between 60-80 breaths/min. The level of isoflurane may be adjusted accordingly to ensure that the animal is fully sedated. With proper monitoring and hydration, animals can be imaged for 4-6 hours, or possibly longer.</li>
	<li>After the imaging experiment, euthanize the animal in a CO2 chamber using IACUC-approved euthanasia protocol. The animal should not be allowed to emerge from anesthesia prior to euthanasia.</li>
</ol>
** This surgical procedure may also be useful for other forms of intravital imaging other than 2P-LSM.
5. Representative Results
Various circulating immune cells are recruited to the LN at different rates following adoptive transfer. For CD4+ and CD8+ lymphocytes, these cells begin to arrive in the LN through the high endothelial venule (HEV) minutes after i.v. transfer with substantial numbers arriving in the popliteal LN after 2 to 4 hours6,17-18. For B cells, a substantial number will accumulate after 8 to 24 hours19. Activated DCs should start to appear in the draining popliteal LN 8 to 16 hours following footpad injection3,11,16-17. Figure 2a shows that even without other landmarks, structures such as B cell follicles can be discerned easily by the round spherical cell accumulation visible under 2P-LSM19. Using an endogenous fluorescent reporter such as the ubiquitin-GFP splenocytes (Figure 2, Supplemental Videos 1 and 2), one can track these lymphocyte migrations and behaviors for days up to a week under non-stimulatory physiologic conditions. With multi-channel high sensitivity detectors, it is possible to acquire a multiplex imaging dataset that encompasses structural information as well as interaction dynamics among multiple cellular partners17,19.
When the surgical techniques are properly executed and the environmental conditions carefully monitored, lymphocytes should exhibit characteristic migration speed, as demonstrated in Figure 2c, 2d and elsewhere13-14,16. Lymphocytes may also exhibit differences in migration speed depending on the sub-regions of the LN undergoing imaging, so additional landmarks such as blood vessels (as highlighted by the introduction of vessel dyes) will help to determine the overall imaging quality (i.e. proper temperature control, minimal trauma to LN, etc.)3,6,20.
<img alt="Figure 1." src="/files/ftp_upload/3720/3720fig1.jpg" /> 
Figure 1. Construction of a mouse holder for mouse popliteal LN Imaging. a) Schematics of mouse holder assembly; b) Representative intravital mouse preparation; c) Completed mouse holder assembly; d) Close-up views of the popliteal LN after surgical exposure.
<img alt="Figure 2." src="/files/ftp_upload/3720/3720fig2.jpg" /> 
Figure 2. Migration analysis of GFP+ lymphocytes in the popliteal LN. (a) 3D snapshot taken from 2P-LSM imaging sequence of the popliteal LN in a C57BL/6 recipient mouse adoptively transferred with 1x107 GFP+ lymphocytes 1 day prior to imaging. Dash line denotes the border of B cell follicles; (b) Tracks of lymphocyte migration during 1 hour of continuous imaging; c) Distribution of overall lymphocyte migration speed. Mean speed = 10.04 &plusmn; 4.26 &mu;m/min (total of 15,125 tracks analyzed); d) Differential cellular migration speed distribution of cells found in the B cell follicle (open bars; mean speed = 8.79 &plusmn; 3.90 &mu;m/min; total 1,525 tracks analyzed) and T cell zone (closed bars; mean speed = 13.77 &plusmn; 5.93 &mu;m/min; total of 1,250 tracks analyzed). Scale bar = 50 &mu;m.
Supplemental Video 1. Time-lapse intravital 2P-LSM imaging of a mouse popliteal LN as described in Figure 2. A total of 1x107 lymphocytes were isolated from an ubiquitin-GFP+ donor mouse and adoptively transferred intravenously into a C57BL/6 recipient mouse 24 hours before imaging. A series of xy (750 &mu;m x 750 &mu;m) fluorescence images were taken through fixed z stacks (5 &mu;m steps, 13 steps) to yield an xyz imaging stack (750 &mu;m x 750 &mu;m x 65 &mu;m), which was repeated every 20 seconds for a total of 60 minutes, resulting in an xyzt imaging sequence for speed analysis (Figures 2c, 2d). Playback speed = 450x. Scale bar = 50 &mu;m. Time stamp = min:sec. <a href="https://www.jove.com/files/ftp_upload/3720/Huang_Jove_video1.avi" target="_blank"> Click here to watch supplemental video</a>.
Supplemental Video 2. Zoomed-in view of the imaging sequence in Supplemental Video 1 at the B cell follicle - T cell zone border. Playback speed = 450x. Scale bar = 25 &mu;m. Time stamp = min: sec. <a href="https://www.jove.com/files/ftp_upload/3720/Huang_Jove_video2.avi" target="_blank"> Click here to watch supplemental video</a>.

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The authors do not have anything to disclose.

Recent advances in high-resolution in situ imaging techniques, especially 2P-LSM, have been accompanied by a growing interest in the study of dynamic cellular behavior in vivo. The 4D imaging technique on the popliteal LN of a live mouse allows such analyses in the dynamic behavior of immune cells within the undisrupted tissue micro-environment. The use of 2P-LSM with multiple detectors spanning the entire visible spectrum permits simultaneous imaging data collection of multiple cell populations. This can now be achieved through the use of in vivo cell-specific fluorescent reporter mice (e.g. Ubiquitin-eGFP, -RFP, or -eCFP) combined with the use of adoptive transfer of differentially labeled cell populations using organic fluorescent cell dyes (e.g. CFSE, SNARF-1, and Cell Tracker Orange) to examine cellular mechanisms and function within the LN. In addition to the direct observation of interactions between differentially tracked cell populations, the multiplex imaging dataset can undergo further analysis with commercially available imaging processing software programs (e.g. Imaris, BitPlane Inc.) to further elucidate cell behavior and function. A broad array of possibilities exists to study cellular interaction mechanisms using these in vivo and in silico techniques.
The main limitation of the experimental approach described here is the technical complexity inherent in the surgery approach. This technique requires rigorous training to become familiar with the relevant anatomy and the precise technical procedures and skills as required by this protocol. Further complicating factors include the difficulty in minimizing tissue damage during LN exploration, optimizing tissue stability during imaging, and preventing thermal and laser injury to the LN before and during imaging experimentation. Perturbation to any of these factors will result in less-than-optimal lymphocyte motility and will therefore interfere with proper interpretation of resulting imaging data analyses.

<table border="1">
 <tbody>
 <tr>
 <td>Reagent</td>
 <td>Company</td>
 <td>Catalogue Number</td>
 </tr>
 <tr>
 <td>Isoflurane, USP</td>
 <td>Baxter Healthcare Corporation</td>
 <td>NDC 10019-773-60</td>
 </tr>
 <tr>
 <td>Vetbond</td>
 <td>3M</td>
 <td>1469SB</td>
 </tr>
 <tr>
 <td>Nair &#x2013; hair removal lotion</td>
 <td>Nair</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>PBS, 1X</td>
 <td>Cellgro</td>
 <td>21-040-CV</td>
 </tr>
 <tr>
 <td>Heating pad </td>
 <td>Watlow</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Heating Pad Controller</td>
 <td>Watlow</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Air and O2</td>
 <td>Airgas</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Temperature probe</td>
 <td>Harvard Apparatus</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Tweezers Dumont #5</td>
 <td>World Precision Instruments, Inc</td>
 <td>14101</td>
 </tr>
 <tr>
 <td>Forceps, Graefe Iris, 7 cm curved</td>
 <td>World Precision Instruments, Inc</td>
 <td>14141</td>
 </tr>
 <tr>
 <td>Scissors</td>
 <td>Roboz Surgical Instrument Co., Inc</td>
 <td>RS-5880</td>
 </tr>
 <tr>
 <td>Cover of 100x20 mm glass cell culture dish</td>
 <td>Corning</td>
 <td>70165-102</td>
 </tr>
 <tr>
 <td>Cover of 100x20 mm polystyrene cell culture dish</td>
 <td>Corning</td>
 <td>430167</td>
 </tr>
 <tr>
 <td>Betadine Solution (10% Povidine-Iodine Topical Solution)</td>
 <td>Purdue Products, L.P.</td>
 <td>NDC 67618-150-08</td>
 </tr>
 </tbody>
 </table>

intravital imaging of the mouse popliteal lymph node

Lymph nodes (LNs) are secondary lymphoid organs, which are strategically located throughout the body to allow for trapping and presentation of foreign antigens from peripheral tissues to prime the adaptive immune response. Juxtaposed between innate and adaptive immune responses, the LN is an ideal site to study immune cell interactions1,2. Lymphocytes (T cells, B cells and NK cells), dendritic cells (DCs), and macrophages comprise the bulk of bone marrow-derived cellular elements of the LN. These cells are strategically positioned in the LN to allow efficient surveillance of self antigens and potential foreign antigens3-5. The process by which lymphocytes successfully encounter cognate antigens is a subject of intense investigation in recent years, and involves an integration of molecular contacts including antigen receptors, adhesion molecules, chemokines, and stromal structures such as the fibro-reticular network2,6-12. 
Prior to the development of high-resolution real-time fluorescent in vivo imaging, investigators relied on static imaging, which only offers answers regarding morphology, position, and architecture. While these questions are fundamental in our understanding of immune cell behavior, the limitations intrinsic with this technique does not permit analysis to decipher lymphocyte trafficking and environmental clues that affect dynamic cell behavior. Recently, the development of intravital two-photon laser scanning microscopy (2P-LSM) has allowed investigators to view the dynamic movements and interactions of individual cells within live LNs in situ12-16. In particular, we and others have applied this technique to image cellular behavior and interactions within the popliteal LN, where its compact, dense nature offers the advantage of multiplex data acquisition over a large tissue area with diverse tissue sub-structures11,17-18. It is important to note that this technique offers added benefits over explanted tissue imaging techniques, which require disruption of blood, lymph flow, and ultimately the cellular dynamics of the system. Additionally, explanted tissues have a very limited window of time in which the tissue remains viable for imaging after explant. With proper hydration and monitoring of the animal's environmental conditions, the imaging time can be significantly extended with this intravital technique. Here, we present a detailed method of preparing mouse popliteal LN for the purpose of performing intravital imaging.

Watch this Scientific Journal Video about Intravital Imaging of the Mouse Popliteal Lymph Node at JoVE.com

Intravital Imaging of the Mouse Popliteal Lymph Node

Recent advances in 2-photon microscopy have enabled real-time in situ imaging of live tissues in animal models, thereby enhancing our ability to investigate cellular behavior in both physiologic and pathologic conditions. Here, we outline the preparations required to perform intravital imaging of the mouse popliteal lymph node.

Lymph nodes (LNs) are secondary lymphoid organs, which are strategically located throughout the body to allow for trapping and presentation of foreign ...

intravital-imaging-of-the-mouse-popliteal-lymph-node

Research

JoVE Journal

Immunology and Infection

28.2K vistas.  Case Western Reserve University. Los recientes avances en microscopía de dos fotones han permitido en tiempo real En Imágenes in situ de los tejidos vivos en modelos animales, lo que mejora nuestra capacidad para investigar el comportamiento celular, tanto en condiciones fisiológicas y patológicas. Aquí, se exponen los trabajos preparatorios necesarios para realizar las imágenes intravital del nodo linfático poplíteo del ratón.

Video: Intravital Imaging of the Mouse Popliteal Lymph Node

Intravital Microscopy of the Inguinal Lymph Node

Lymph nodes (LN's), located throughout the body, are an integral component of the immune system. They serve as a site for induction of adaptive immune response and therefore, the development of effector cells. As such, LNs are key to fighting invading pathogens and maintaining health. The choice of LN to study is dictated by accessibility and the desired model; the inguinal lymph node is well situated and easily supports studies of biologically relevant models of skin and genital mucosal infection.
The inguinal LN, like all LNs, has an extensive microvascular network supplying it with blood. In general, this microvascular network includes the main feed arteriole of the LN that subsequently branches and feeds high endothelial venules (HEVs). HEVs are specialized for facilitating the trafficking of immune cells into the LN during both homeostasis and infection. How HEVs regulate trafficking into the LN under both of these circumstances is an area of intense exploration. The LN feed arteriole, has direct upstream influence on the HEVs and is the main supply of nutrients and cell rich blood into the LN. Furthermore, changes in the feed arteriole are implicated in facilitating induction of adaptive immune response. The LN microvasculature has obvious importance in maintaining an optimal blood supply to the LN and regulating immune cell influx into the LN, which are crucial elements in proper LN function and subsequently immune response. 
The ability to study the LN microvasculature in vivo is key to elucidating how the immune system and the microvasculature interact and influence one another within the LN. Here, we present a method for in vivo imaging of the inguinal lymph node. We focus on imaging of the microvasculature of the LN, paying particular attention to methods that ensure the study of healthy vessels, the ability to maintain imaging of viable vessels over a number of hours, and quantification of vessel magnitude. Methods for perfusion of the microvasculature with vasoactive drugs as well as the potential to trace and quantify cellular traffic are also presented. 
Intravital microscopy of the inguinal LN allows direct evaluation of microvascular functionality and real-time interface of the direct interface between immune cells, the LN, and the microcirculation. This technique potential to be combined with many immunological techniques and fluorescent cell labelling as well as manipulated to study vasculature of other LNs.

A technique for performing intravital microscopy of the inguinal lymph node (LN) is outlined. Such technique allows for real-time, in vivo study of the lymph node microvasculature and structure both during homeostasis and infection. This technique can be adapted to cell trafficking studies and to other lymph node sites.

Lymph nodes (LN's), located throughout the body, are an integral component of the immune system. They serve as a site for induction of adaptive immune ...

Ex vivo Imaging of T Cells in Murine Lymph Node Slices with Widefield and Confocal Microscopes

Na&iuml;ve T cells continuously traffic to secondary lymphoid organs, including peripheral lymph nodes, to detect rare expressed antigens. The migration of T cells into lymph nodes is a complex process which involves both cellular and chemical factors including chemokines. Recently, the use of two-photon microscopy has permitted to track T cells in intact lymph nodes and to derive some quantitative information on their behavior and their interactions with other cells. While there are obvious advantages to an in vivo system, this approach requires a complex and expensive instrumentation and provides limited access to the tissue. To analyze the behavior of T cells within murine lymph nodes, we have developed a slice assay 
1, originally set up by neurobiologists and transposed recently to murine thymus 2. In this technique, fluorescently labeled T cells are plated on top of an acutely prepared lymph node slice. In this video-article, the localization and migration of T cells into the tissue are analyzed in real-time with a widefield and a confocal microscope. The technique which complements in vivo two-photon microscopy offers an effective approach to image T cells in their natural environment and to elucidate mechanisms underlying T cell migration.

Ex vivo Imaging of T Cells in Murine Lymph Node Slices with Widefield and Confocal Microscopes

This protocol describes a method to image fluorescent T cells introduced into lymph node slices. The technique permits real-time analyses of T cell migration with traditional widefield fluorescence or confocal microscopes.

Na&iuml;ve T cells continuously traffic to secondary lymphoid organs, including peripheral lymph nodes, to detect rare expressed antigens. The migration ...

Murine Superficial Lymph Node Surgery

In the field of immunology, to understand the progression of an immune response against a vaccine, an infection or a tumour, the response is often followed over time. Similarly, the study of lymphocyte homeostasis requires time course experiments. Performing these studies within the same mouse is ideal to reduce the experimental variability as well as the number of mice used. Blood withdrawal allows performance of time course experiments, but it only gives information about circulating lymphocytes and provides a limited number of cells1-4. Since lymphocytes circulating through the body and residing in the lymph nodes have different properties, it is important to examine both locations. The sequential removal of lymph nodes by surgery provides a unique opportunity to follow an immune response or immune cell expansion in the same mouse over time. Furthermore, this technique yields between 1-2x106 cells per lymph node which is sufficient to perform phenotypic characterization and/or functional assays. Sequential lymph node surgery or lymphadenectomy has been successfully used by us and others5-11. Here, we describe how the brachial and inguinal lymph nodes can be removed by making a small incision in the skin of an anesthetised mouse. Since the surgery is superficial and done rapidly, the mouse recovers very quickly, heals well and does not experience excessive pain. Every second day, it is possible to harvest one or two lymph nodes allowing for time course experiments. This technique is thus suitable to study the characteristics of lymph node-residing lymphocytes over time. This approach is suitable to various experimental designs and we believe that many laboratories would benefit from performing sequential lymph node surgeries.

To follow the progression of an immune response over time within the same mouse, lymph nodes can be sequentially removed by surgery. Here, we describe how this technique can be performed.

In the field of immunology, to understand the progression of an immune response against a vaccine, an infection or a tumour, the response is often ...

Intravital Imaging of the Mouse Thymus using 2-Photon Microscopy

Two-photon Microscopy (TPM) provides image acquisition in deep areas inside tissues and organs. In combination with the development of new stereotactic tools and surgical procedures, TPM becomes a powerful technique to identify "niches" inside organs and to document cellular "behaviors" in live animals. While intravital imaging provides information that best resembles the real cellular behavior inside the organ, it is both more laborious and technically demanding in terms of required equipment/procedures than alternative ex vivo imaging acquisition. Thus, we describe a surgical procedure and novel "stereotactic" organ holder that allows us to follow the movements of Foxp3+ cells within the thymus.
Foxp3 is the master regulator for the generation of regulatory T cells (Tregs). Moreover, these cells can be classified according to their origin: ie. thymus-differentiated Tregs are called "naturally-occurring Tregs" (nTregs), as opposed to peripherally-converted Tregs (pTregs). Although significant amount of research has been reported in the literature concerning the phenotype and physiology of these T cells, very little is known about their in vivo interactions with other cells. This deficiency may be due to the absence of techniques that would permit such observations. The protocol described in this paper provides a remedy for this situation.
Our protocol consists of using nude mice that lack an endogenous thymus since they have a punctual mutation in the DNA sequence that compromises the differentiation of some epithelial cells, including thymic epithelial cells. Nude mice were gamma-irradiated and reconstituted with bone marrows (BM) from Foxp3-KIgfp/gfp mice. After BM recovery (6 weeks), each animal received embryonic thymus transplantation inside the kidney capsule. After thymus acceptance (6 weeks), the animals were anesthetized; the kidney containing the transplanted thymus was exposed, fixed in our organ holder, and kept under physiological conditions for in vivo imaging by TPM. We have been using this approach to study the influence of drugs in the generation of regulatory T cells.

We have developed novel laboratory tools and protocols for intravital imaging acquisition of the thymus. Our technique should help in the identification of &#x201C;niches&#x201D; within the thymus where T cell development occurs.

Two-photon Microscopy (TPM) provides image acquisition in deep areas inside tissues and organs. In combination with the development of new stereotactic ...

Generation of Lymph Node-fat Pad Chimeras for the Study of Lymph Node Stromal Cell Origin

The stroma is a key component of the lymph node structure and function. However, little is known about its origin, exact cellular composition and the mechanisms governing its formation. Lymph nodes are always encapsulated in adipose tissue and we recently demonstrated the importance of this relation for the formation of lymph node stroma. Adipocyte precursor cells migrate into the lymph node during its development and upon engagement of the Lymphotoxin-b receptor switch off adipogenesis and differentiate into lymphoid stromal cells (B&#233;n&#233;zech&#160;et al.14). Based on the lymphoid stroma potential of adipose tissue, we present a method using a lymph node/fat pad chimera that allows the lineage tracing of lymph node stromal cell precursors. We show how to isolate newborn lymph nodes and EYFP+ embryonic adipose tissue and make a LN/ EYFP+ fat pad chimera. After transfer under the kidney capsule of a host mouse, the lymph node incorporates local adipose tissue precursor cells and finishes its formation. Progeny analysis of EYFP+ fat pad cells in the resulting lymph nodes can be performed by flow-cytometric analysis of enzymatically digested lymph nodes or by immunofluorescence analysis of lymph nodes cryosections. By using fat pads from different knockout mouse models, this method will provide an efficient way of analyzing the origin of the different lymph node stromal cell populations.

Generation of lymph node/fat pad chimeras for the study of lymph node stromal cell origin is described. The method involves the isolation of lymph nodes from newborn mice and embryonic fat pads, the generation of chimeric lymph node-fat pads, and their transfer under the kidney capsule of a host mouse.

The stroma is a key component of the lymph node structure and function. However, little is known about its origin, exact cellular composition and the ...

Highly Resolved Intravital Striped-illumination Microscopy of Germinal Centers

Monitoring cellular communication by intravital deep-tissue multi-photon microscopy is the key for understanding the fate of immune cells within thick tissue samples and organs in health and disease. By controlling the scanning pattern in multi-photon microscopy and applying appropriate numerical algorithms, we developed a striped-illumination approach, which enabled us to achieve 3-fold better axial resolution and improved signal-to-noise ratio, i.e. contrast, in more than 100 &#181;m tissue depth within highly scattering tissue of lymphoid organs as compared to standard multi-photon microscopy. The acquisition speed as well as photobleaching and photodamage effects were similar to standard photo-multiplier-based technique, whereas the imaging depth was slightly lower due to the use of field detectors. By using the striped-illumination approach, we are able to observe the dynamics of immune complex deposits on secondary follicular dendritic cells &#8211; on the level of a few protein molecules in germinal centers.

High-resolution intravital imaging with enhanced contrast up to 120 &#181;m depth in lymph nodes of adult mice is achieved by spatially modulating the excitation pattern of a multi-focal two-photon microscope. In 100 &#181;m depth we measured resolutions of 487 nm (lateral) and 551 nm (axial), thus circumventing scattering and diffraction limits.

Monitoring cellular communication by intravital deep-tissue multi-photon microscopy is the key for understanding the fate of immune cells within thick ...

Isolation of Murine Lymph Node Stromal Cells

Secondary lymphoid organs including lymph nodes are composed of stromal cells that provide a structural environment for homeostasis, activation and differentiation of lymphocytes. Various stromal cell subsets have been identified by the expression of the adhesion molecule CD31 and glycoprotein podoplanin (gp38), T zone reticular cells or fibroblastic reticular cells, lymphatic endothelial cells, blood endothelial cells and FRC-like pericytes within the double negative cell population. For all populations different functions are described including, separation and lining of different compartments, attraction of and interaction with different cell types, filtration of the draining fluidics and contraction of the lymphatic vessels. In the last years, different groups have described an additional role of stromal cells in orchestrating and regulating cytotoxic T cell responses potentially dangerous for the host. 
Lymph nodes are complex structures with many different cell types and therefore require a appropriate procedure for isolation of the desired cell populations. Currently, protocols for the isolation of lymph node stromal cells rely on enzymatic digestion with varying incubation times; however, stromal cells and their surface molecules are sensitive to these enzymes, which results in loss of surface marker expression and cell death. Here a short enzymatic digestion protocol combined with automated mechanical disruption to obtain viable single cells suspension of lymph node stromal cells maintaining their surface molecule expression is proposed.

Isolation of lymph node stromal cells is a multistep procedure including enzymatic digestion and mechanical disaggregation to obtain fibroblastic reticular cells, lymphatic and blood endothelial cells. In the described procedure, a short digestion is combined with automated mechanical disaggregation to minimize surface marker degradation of viable lymph node stromal cells.

Secondary lymphoid organs including lymph nodes are composed of stromal cells that provide a structural environment for homeostasis, activation and ...

Intravital Microscopy Imaging of the Liver following Leishmania Infection: An Assessment of Hepatic Hemodynamics

Intravital microscopy (IVM) is a powerful optical imaging technique that has made possible the visualization, monitoring and quantification of various biological events in real time and in live animals. This technology has greatly advanced our understanding of physiological processes and pathogen-mediated phenomena in specific organs.
In this study, IVM is applied to the mouse liver and protocols are designed to image in vivo the circulatory system of the liver and measure red blood cell (RBC) velocity in individual hepatic vessels. To visualize the different vessel subtypes that characterize the hepatic organ and perform blood flow speed measurements, C57Bl/6 mice are intravenously injected with a fluorescent plasma reagent that labels the liver-associated vasculature. IVM enables in vivo, real time, measurement of RBC velocity in a specific vessel of interest. Establishing this methodology will make it possible to investigate liver hemodynamics under physiological and pathological conditions. Ultimately, this imaging-based methodology will be important for studying the influence of L. donovani infection&#160;on hepatic hemodynamics.
This method can be applied to other infectious models and mouse organs and might be further extended to pre-clinical testing of a drug&#8217;s effect on inflammation by quantifying its effect on blood flow.

Intravital Microscopy Imaging of the Liver following Leishmania Infection: An Assessment of Hepatic Hemodynamics

This article reports on a detailed method for the dynamic measurement and quantification of blood flow velocity within individual blood vessels of the mouse liver vasculature using intravital microscopy imaging in combination with a specific methodology for image acquisition and analysis.

Intravital microscopy (IVM) is a powerful optical imaging technique that has made possible the visualization, monitoring and quantification of various ...

Myeloid Cell Isolation from Mouse Skin and Draining Lymph Node Following Intradermal Immunization with Live Attenuated Plasmodium Sporozoites

Malaria infection begins when the sporozoite stage of Plasmodium is inoculated into the skin of a mammalian host through a mosquito bite. The highly motile parasite not only reaches the liver to invade hepatocytes and transform into erythrocyte-infective form. It also migrates into the skin and to the proximal lymph node draining the injection site, where it can be recognized and degraded by resident and/or recruited myeloid cells. Intravital imaging reported the early recruitment of brightly fluorescent Lys-GFP positive leukocytes in the skin and the interactions between sporozoites and CD11c+ cells in the draining lymph node. We present here an efficient procedure to recover, identify and enumerate the myeloid cell subsets that are recruited to the mouse skin and draining lymph node following intradermal injection of immunizing doses of sporozoites in a murine model. Phenotypic characterization using multi-parametric flow cytometry provides a reliable assay to assess early dynamic cellular changes during inflammatory response to Plasmodium infection.

Myeloid Cell Isolation from Mouse Skin and Draining Lymph Node Following Intradermal Immunization with Live Attenuated Plasmodium Sporozoites

We describe here a protocol for isolating myeloid cells from mouse skin and draining lymph node following intradermal injection of Plasmodium sporozoites. Flow cytometry of collected cells provides a reliable assay to characterize the skin and draining lymph node inflammatory response to the parasite.

Malaria infection begins when the sporozoite stage of Plasmodium is inoculated into the skin of a mammalian host through a mosquito bite. The highly ...

Two-photon Intravital Imaging of Leukocytes During the Immune Response in Lipopolysaccharide-treated Mouse Liver

Sepsis is a type of severe infection that can cause organ failure and tissue damage. Although the mortality and morbidity rates associated with sepsis are extremely high, no direct treatment or organ-related mechanism has been examined in detail in real time. The liver is the key organ that manages toxins and infections in the human body. Herein, we aimed to perform intravital imaging of mouse liver after induction of endotoxemia in order to track the motility of immune cells, such as neutrophils and liver capsular macrophages (LCMs). Accordingly, we designed a novel surgical method for exposure of the liver with minimally invasive surgery. Mice were intraperitoneally injected with lipopolysaccharide (LPS), a common endotoxin. Using our novel surgical approach for exposure and intravital imaging of the mouse liver, we found that neutrophil recruitment in LPS-treated LysM-green fluorescent protein (GFP) mouse liver was increased compared with that in phosphate-buffered saline-treated liver. After LPS treatment, the number of neutrophils increased significantly with time. Additionally, using CX3Cr1-GFP mice, we successfully visualized liver resident macrophages called LCMs. Therefore, to investigate the efficacy of new reagents to control immune mobility in vivo, determining the motility and morphology of neutrophils and LCMs in the liver may allow us to identify therapeutic effect in organ failure and tissue damage caused by leukocytes activation in sepsis.

We established a novel surgical protocol for two-photon imaging of live mice liver with minimal invasion. With this technique, we identified the detailed structure of the liver during lipopolysaccharide-induced endotoxemia. We anticipate that this method may be utilized to determine the effectiveness of various reagents treatment to hepatic leukocyte migration.

Sepsis is a type of severe infection that can cause organ failure and tissue damage. Although the mortality and morbidity rates associated with sepsis are ...