This work is funded by the Canadian Institutes of Health Research, Canadian Foundation for Innovation, University of British Columbia Centre for Blood Research and University of Northern BC.

1. Reagent Preparation
<ol>
<li>Reagents to be used throughout the experiment should be prepared before starting the surgical protocol. Note that all solutions were made using sterile technique.</li>
<li>Physiological saline solution (PSS) is best prepared in two components: basic salt solution and sodium bicarbonate solution. Prepare both solutions at 20X concentration and store at 4&deg;C. Sodium bicarbonate at 20X is 360.0mM NaHCO3 (84.01g/mol) and basic salt solution at 20X concentration is 2638.0mM NaCl (58.44g/mol), 94.0mM KCl (74.56g/mol), 40.0mM CaCl2&bull;2H2O (147.02g/mol), and 23.4mM MgSO4&bull;7H2O (246.48g/mol). Solutions should be prepared in dH2O and chemicals should be added one at a time and stirred until solution is clear prior to adding the next chemical</li>
<li>Prepare 2L of PSS by diluting equal volumes of sodium bicarbonate and basic salt solution and diluting to 1X concentration (for two 2L of PSS dilute 100mL of each sodium bicarbonate and basic salt solution in dH2O</li>
<li>Place 2L volumetric flask containing PSS and PSS in 37&deg;C water bath. Equilibrate the solution with 5% CO2 / balance nitrogen gas for 1hr prior to start of surgical procedure.</li>
<li>Vasoactive chemicals (ex. Phenylephrine, acetylcholine, sodium nitroprusside) are best prepared at 1M concentration in dH2O and stored in 100&mu;l aliquots at -20&deg;C.</li> 
</ol>
2. Animal Preparation
<ol>
<li>Determine the weight of the mouse and administer the initial dose of sodium pentobarbital at 90mg/kg by intraperitoneal injection. Throughout the procedure the mouse is continually monitored for anesthesia plane via toe pinch and careful examination of respiratory rate. Additional injections of sodium pentobarbital should be administered as needed at 20mg/kg to maintain anesthesia plane. The surgical plane of anesthesia is maintained in accordance with federal and institutional regulations as outlined in the animal care protocol approved by the Animal Care and Use Committee (ACUC) of the University of Northern BC.</li>
<li>Remove the hair from the abdominal and flank/hip area by shaver. Remove remaining loose hair by alcohol swab and patting the area with tape. This is sufficient as this experimental procedure is terminal.</li>
<li>Place the mouse on a clear surgical board in a supine position and attach the mouse to the board by taping each footpad to the board. Body temperature is maintained via an external heat lamp and monitored via rectal thermometer to ensure that hypothermia does not occur as a result of anesthetic.</li> 
</ol>
3. Surgical Procedure
<ol>
<li>During the entire surgical procedure ensure exposed tissue is always kept superfused with equilibrated warmed PSS solution. It is also important to never make contact with the vasculature of interest with surgical instruments or injure the vessels by placing to much force on them by manipulating adipose tissue and connective tissue around them.</li>
<li>Place the mouse under the dissecting scope. Make a midline incision along the ventral surface of the abdominal cavity and retract the skin towards the mouse's spinal column.</li> 
<li>Once the skin is retracted so that the LN is easily visible, pin the skin onto a pedestal of transparent Sylgard 184.</li> 
<li>Remove the thin layer of connective tissue overlaying the area around the LN</li> 
<li>Clear the adipose tissue overlaying and surround the lymph node until the microvascular bed is exposed. The main arterial segment lays adjacent to the LN and is commonly overlayed by the venule. The entire surgery takes approximately 30 minutes.</li>
</ol>
4. Imaging and Vessel Analysis
<ol>
<li>Once the vessel segment of interest is exposed under the dissecting scope, secure the preparation on the intravital microscope. Note that animal remains on the clear surgical board while on the intravital microscope throughout the duration of the experiment. The body temperature is maintained via heat lamp and measured via rectal thermometer as described during the surgical section. The anesthesia plane is also monitored throughout the experiment as described in section 2.1.</li>
<li>Set up the superfusion and suction lines. Superfusion of PSS occurs by gravity feed from a reservoir, into the water jacket heated reservoir (the water jacket is circulated and heated by the circulating waterbath) being perfused by 5% CO2 balanced nitrogen, and down a drip line at a rate of 10ml/min. A suction line should be used to continue pull superfused PSS away from the preparation. Note that prior to the experiment the suction lines and water jacket are thoroughly cleaned and stored after each experiment. This ensures sterility prior to the next experiment.</li>
<li>Check the temperature of the superfusing PSS. Adjust the temperature of the circulating water bath and/or the rate of PSS superfusion to achieve a temperature of &tilde;37&deg;C across the preparation.</li>
<li>Allow the vasculature to equilibrate with PSS for at least 60 minutes and quantify the vessel diameter using the video caliper. Typically, vessel diameter should be determined at multiple points (for example, at 40, 50, and 60 minutes) to ensure the vessel has stabilized.</li> 
<li>Assess the health of the vessel using a smooth muscle specific agonist (ex. Phenylephrine, PE) and an endothelium specific agonist (ex. Acetylcholine, ACh). Concentration of agonist should be adjusted depending on the agonist, but for PE and Ach the vessel should be evaluated at each concentration by cumulative addition (10-5M to 10-9M) to the superfusate. Each agonist application should be separated by a wash phase (typically 30 minutes) using PSS until the vessel returns to resting diameter.</li>
<li>Following evaluation of vessel health, subsequent vasoactive drugs, florescent labeling, and cell tracing experiments can be conducted.</li>
<li>Following the end of the experiment the animal is given an overdose of sodium pentobarbital. The animal is then monitored to ensure that there is no response to toe pinch and lack of respiratory movement or heart beat. At such time this is followed by cervical dislocation.</li> 
</ol>
5. Representative Results:
Following the equilibration period the preparation should yield a clear image that allows for identification of both the arteriole and venule that supplies the inguinal lymph node. There should be minimal adipose cells surrounding the vessels to allow for walls of the arteriole and venule to be clearly observed for the video calipers to be superimposed to calculate vessel diameter. The integral point of a successful preparation is the arteriole has a rich supply of blood moving through the lumen and that there is a significant degree of vascular tone (i.e. the arteriole has the ability to vasoconstrict and vasodilate to smooth muscle and endothelial specific agonists respectively).
<img src="/files/ftp_upload/2551/2551fig1.jpg" alt="Figure 1"/> Figure 1. Intravital microscopy image of inguinal lymph node feed arteriole (red arrow) feeding the lymph node equilibrated with physiological saline solution and running along side the main venule (green arrow).
<img src="/files/ftp_upload/2551/2551fig2.jpg" alt="Figure 2"/> Figure 2. Normal vasoactive response to superfused phenylephrine (PE) by cumulative addition to physiological saline from the concentrations 10-9M to 10-5M. The presence of a robust vasoconstriction is suggestive of normal smooth muscle function present in the arteriole.
<img src="/files/ftp_upload/2551/2551fig3.jpg" alt="Figure 3"/> Figure 3. Normal vasoactive response to superfused acetylcholine (ACh) by cumulative addition to physiological saline from concentrations 10-9M to 10-5M. The presence of a robust vasodilation is suggestive of normal endothelial function present in the arteriole.

<ol>
	<li>Bearden, S. E., Payne, G. W., Chisty, A., Segal, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arteriolar+network+architecture+and+vasomotor+function+with+ageing+in+mouse+gluteus+maximus+muscle.">Arteriolar network architecture and vasomotor function with ageing in mouse gluteus maximus muscle.</a> J Physiol. 561, 535-545 (2004).</li><li>Looft-Wilson, R. C., Payne, G. W., Segal, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Connexin+expression+and+conducted+vasodilation+along+arteriolar+endothelium+in+mouse+skeletal+muscle.">Connexin expression and conducted vasodilation along arteriolar endothelium in mouse skeletal muscle.</a> J Appl Physiol. 97, 1152-1158 (2004).</li><li>Payne, G. W., Madri, J. A., Sessa, W. C., Segal, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histamine+inhibits+conducted+vasodilation+through+endothelium-derived+NO+production+in+arterioles+of+mouse+skeletal+muscle.">Histamine inhibits conducted vasodilation through endothelium-derived NO production in arterioles of mouse skeletal muscle.</a> Faseb J. 18, 280-286 (2004).</li><li>Smeda, J. S., Payne, G. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alterations+in+autoregulatory+and+myogenic+function+in+the+cerebrovasculature+of+Dahl+salt-sensitive+rats.">Alterations in autoregulatory and myogenic function in the cerebrovasculature of Dahl salt-sensitive rats.</a> Stroke. 34, 1484-1490 (2003).</li><li>de With, M. C., de Vries, A. M., Kroese, A. B., van der Heijden, E. P., Bleys, R. L., Segal, S. S., Kon, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+anatomy+of+the+hamster+retractor+muscle+with+regard+to+its+microvascular+transfer.">Vascular anatomy of the hamster retractor muscle with regard to its microvascular transfer.</a> Eur Surg Res. 42, 97-105 (2009).</li><li>Domeier, T. L., Segal, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electromechanical+and+pharmacomechanical+signalling+pathways+for+conducted+vasodilatation+along+endothelium+of+hamster+feed+arteries.">Electromechanical and pharmacomechanical signalling pathways for conducted vasodilatation along endothelium of hamster feed arteries.</a> J Physiol. 579, 175-186 (2007).</li></ol>

No conflicts of interest declared.

Intravital microscopy of the inguinal lymph node presented here provides the ability to image microvasculature of the lymph node in-vivo. Thus, it facilitates a means of direct, real-time observation. Imaging of the LN microvasculature is a unique site that allows one to study the interface between the immune response and the vasculature. Using this preparation, focus can be directed specifically at the immune response, alterations in the vasculature, or at interaction between the two. 
As with all experimental approaches, standard intravital microscopy has both advantages and limitations. Standard IVM, such as the preparation described here, can easily be modified, and has been previously demonstrated by the authors to allow epifluorescent microscopy via the introduction of fluorescent tracer dyes or labeled cell populations. Although standard IVM does not give the possibility of three dimensional imaging and tracing such as would be given by two-photon microscopy or angiography, cell tracking is still achieved in two dimensions allowing cell-to-cell and cell-to-vasculature interaction to be observed and quantified and in conjunction with in-vivo administration and subsequent staining with fluorescent antibodies can be used to give data on protein/marker expression in real-time.
Notably, alternative imaging techniques require a static environment for images; clamping of the surgical area or the addition of a coverslip on a flat preparation is frequently needed. This limits, if not eliminates, the ability to actively perfuse vascular or other mediators over the preparation to evaluate vascular integrity and physiology etc. or the use of other techniques such as conducted vasodilation experiments within the IVM preparation. Furthermore, standard IVM does not require a completely flat preparation and is not affected by motion, such as that generated by the breathing of the animal. This allows standard IVM to be used in more surgical areas with greater reproducibility. This is exemplified by the inguinal lymph node preparation described here. Given the size and shape of the lymph node, the preparation can not be made flat without injuring the tissue and the location of the node gives rise to significant motion due to animal respiration. Such issues would be difficult to overcome by other methods, but are easily dealt with using the standard IVM preparation described. 
In summary, the preparation detailed above can be combined with any number of other biochemical, vascular, and/or immunological techniques such as transfer of subsets of activated or labeled cells, induction of hypoxia, and over-expression of depletion of vascular mediators. However, additional applications are dependent on the health of the initial preparation. Therefore, it is vital to always test the health of the vasculature being imaged and evaluated. Key points to achieving a health preparation are ensuring the preparation is constantly perfused with equilibrated PSS at body temperature and minimizing contact and stress placed on the surgical area during surgery.

<table border="1">
	<tbody>
 	
 	<tr>
 	<th>Name of the reagent</th>
 <th>Company</th>
 </tr>
 <tr>
 	<td>Sodium Bicarbonate</td>
 <td>Sigma Aldrich</td>
 </tr>

 <tr>
 	<td>Sodium Chloride</td>
 <td>Sigma Aldrich</td>
 </tr>

 <tr>
 	<td>Potassium Chloride</td>
 <td>Sigma Aldrich</td>
 </tr>

 <tr>
 	<td>Calcium Chloride Dihydrate</td>
 <td>Sigma Aldrich</td>
 </tr>

 <tr>
 	<td>Sodium Nitroprusside</td>
 <td>Sigma Aldrich</td>
 </tr>

 <tr>
 	<td>Phenylephrine</td>
 <td>Sigma Aldrich</td>
 </tr>

 <tr>
 	<td>Acetylcholine</td>
 <td>Sigma Aldrich</td>
 </tr>

 <tr>
 	<td>Magnesium chloride heptahydrate</td>
 <td>Sigma Aldrich</td>
 </tr>

 <tr>
 	<td>Sodium Pentobarbitol</td>
 <td>&nbsp;</td>
 </tr> 
 </tbody>
</table>

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.

Watch this Scientific Journal Video about Intravital Microscopy of the Inguinal Lymph Node at JoVE.com

Intravital Microscopy of the Inguinal Lymph Node

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 ...

intravital-microscopy-of-the-inguinal-lymph-node

Research

JoVE Journal

Immunology and Infection

20.0K Views.  University of Northern British Columbia. 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.

Video: Intravital Microscopy of the Inguinal Lymph Node

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 ...

Intravital Microscopy of the Spleen: Quantitative Analysis of Parasite Mobility and Blood Flow

The advent of intravital microscopy in experimental rodent malaria models has allowed major advances to the knowledge of parasite-host interactions 1,2. Thus, in vivo imaging of malaria parasites during pre-erythrocytic stages have revealed the active entrance of parasites into skin lymph nodes 3, the complete development of the parasite in the skin 4, and the formation of a hepatocyte-derived merosome to assure migration and release of merozoites into the blood stream 5. Moreover, the development of individual parasites in erythrocytes has been recently documented using 4D imaging and challenged our current view on protein export in malaria 6. Thus, intravital imaging has radically changed our view on key events in Plasmodium development. Unfortunately, studies of the dynamic passage of malaria parasites through the spleen, a major lymphoid organ exquisitely adapted to clear infected red blood cells are lacking due to technical constraints.
Using the murine model of malaria Plasmodium yoelii in Balb/c mice, we have implemented intravital imaging of the spleen and reported a differential remodeling of it and adherence of parasitized red blood cells (pRBCs) to barrier cells of fibroblastic origin in the red pulp during infection with the non-lethal parasite line P.yoelii 17X as opposed to infections with the P.yoelii 17XL lethal parasite line 7. To reach these conclusions, a specific methodology using ImageJ free software was developed to enable characterization of the fast three-dimensional movement of single-pRBCs. Results obtained with this protocol allow determining velocity, directionality and residence time of parasites in the spleen, all parameters addressing adherence in vivo. In addition, we report the methodology for blood flow quantification using intravital microscopy and the use of different colouring agents to gain insight into the complex microcirculatory structure of the spleen. 
Ethics statement
All the animal studies were performed at the animal facilities of University of Barcelona in accordance with guidelines and protocols approved by the Ethics Committee for Animal Experimentation of the University of Barcelona CEEA-UB (Protocol No DMAH: 5429). Female Balb/c mice of 6-8 weeks of age were obtained from Charles River Laboratories.

We show the method for performing intravital microscopy of the spleen using GFP transgenic malaria parasites and the quantification of parasite mobility and blood flow within this organ.

The advent of intravital microscopy in experimental rodent malaria models has allowed major advances to the knowledge of parasite-host interactions 1,2. ...

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.

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 ...

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 ...

Intravital Microscopy of Leukocyte-endothelial and Platelet-leukocyte Interactions in Mesenterial Veins in Mice

Intravital microscopy is a method that can be used to investigate different processes in different regions and vessels in living animals. In this protocol, we describe intravital microscopy of mesentery veins. This can be performed in a short period of time with reproducible results showing leukocyte-endothelial interactions in vivo. We describe an inflammatory setting after LPS challenge of the endothelium. But in this model one can apply many different types of inflammatory conditions, like bacterial, chemical or biological and investigate the administration of drugs and their direct effects on the living animal and its impact on leukocyte recruitment. This protocol has been applied successfully to a number of different treatments of mice and their effects on inflammatory response in vessels. Herein, we describe the visualization of leukocytes and platelets by fluorescently labeling these with rhodamine 6G. Additionally, any specific imaging can be performed using targeted fluorescently labeled molecules.

This simplified application of intravital microscopy of mesentery veins in mice can be used in different models of inflammation to observe leukocyte-endothelial and platelet-leukocyte interactions.