Name: Visualizing Lymph Node Structure and Cellular Localization using Ex-Vivo Confocal Microscopy (Video) | JoVE Journal
Uploaded: 2019-08-09T13:30:00.000+00:00
Duration: 7 min 40 s
Description: Learn ex-vivo confocal microscopy for visualizing lymph node structure and cellular localization using fluorescent labeled antibodies in mouse models. This method preserves tissue integrity while enabling specific visualization of T and B cell distribution.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

This work was supported by the NIH (R01 AI43458 to H.L.W.).

The protocol was approved by the Standing Committee on Animals at Harvard Medical School and Brigham and Women&#8217;s Hospital, protocol 2016N000230.
1. Mice used for the experiment
<ol>
	<li>Use 8-week old male and female mice on the B6 background for administering the antibody mix.</li>
	<li>Use CX3CR1GFP/WTCCR2RFP/WT mice to determine whether ex vivo whole LN imaging can also be applied to reporter mice without administering antibody mix as well as to investigate the presence of mononuclear cells, including antigen presenting cells and phagocytes, and their distribution in the LN. 
	NOTE: CX3CR1GFP/WTCCR2RFP/WT reporter mice have green fluorescent protein (GFP) and red fluorescent protein (RFP) inserted under the control of CX3CR1 and CCR2 promoters, respectively. Reporter mice can be used with or without the injection of the antibody mix. Please see reference4 for antibody mix injection in a reporter mouse. Proceed to surgery if no antibody will be injected.</li>
</ol>
2. Antibody mix preparation and injection
NOTE: Perform these steps on mice described in step 1.1.
<ol>
	<li>Dilute 1:10 of brilliant violet (BV) 421 anti-CD4 (GK1.5; 0.2 mg/mL), 1:10 of brilliant blue (BB) 515 anti-CD19 (1D3; 0.2 mg/mL) and 1:20 of phycoerythrin (PE) anti-F4/80 (T45-2342; 0.2 mg/mL) in PBS with the appropriate final volume to inject into the inner thigh (to image inguinal) or into the paw pad (to image popliteal) lymph. 
	NOTE: Use as isotypes: BV421 Mouse IgG2b, k Isotype Control (R35-38; 0.2 mg/mL); PE Rat IgG2a, &#954; Isotype Control (R35-95; 0.2 mg/mL); BB515 Rat IgG2a, &#954; Isotype Control (R35-95; 0.2 mg/mL). If changing the staining antibody, use correct isotype.</li>
	<li>To image inguinal LN, inject 100 &#181;L of the antibody mix subcutaneously into the inner thigh (Figure 1A). Alternatively, inject 50 &#181;L subcutaneously of antibody mix into the paw pad to image popliteal LN (Figure 2A). Use delicate 1 mL Insulin syringes, (Insulin U-100) with the needle of size 0.30 mm &#215; 13 mm (30 gauge &#215; &#189; inch). 
	NOTE: Ensure that the injection for inguinal LN staining is subcutaneous and not intramuscular (i.m.), as antibody mix will not be properly drained if i.m. administration is performed.</li>
	<li>Do not anesthetize animals before antibody mix injection.</li>
	<li>Wait for a minimum of 3 h (inguinal dLN) and 12 h (popliteal dLN) post injection to remove the organs.</li>
	<li>If LN cell labeling is not completely observed using large polymer fluorescent dyes such as brilliant violet or brilliant blue, use smaller fluorophores, including fluorescein isothiocyanate (FITC), PE and allophycocyanin (APC) as an alternative.</li>
</ol>
3. Surgery procedure to remove the inguinal draining lymph node
<ol>
	<li>Euthanize mice using CO2 asphyxiation followed by cervical dislocation.</li>
	<li>Immobilize mice on the acrylic stage with adhesive tape and apply mineral oil with a cotton swab to the abdominal skin to prevent fur deposition around the incision (Figure 1B). Fur removal is not necessary.</li>
	<li>Perform a midline incision using microsurgery curved scissors (11.5 cm) and microsurgery curved forceps (12.5 cm) in the abdomen from the pubis to the xiphoid process (Figure 1C).</li>
	<li>Dissociate the abdominal musculature from the skin (Figure 1D).</li>
	<li>Make horizontal skin incisions at the top and bottom of the vertical incision line to create skin flaps on side of interest (according to the side of the antibody mix injection) and flap the skin to visualize the lymph node (Figure 1E).</li>
	<li>Tape the skin-flap on the acrylic plate (Figure 1F).</li>
	<li>Remove the inguinal draining lymph node using microsurgery curved forceps (Figure 1F). Lymph node will appear as a translucid, usually bilobular, sphere under the skin.</li>
</ol>
4. Surgery procedure to remove the popliteal draining lymph node
<ol>
	<li>Euthanize mice using CO2 asphyxiation followed by cervical dislocation.</li>
	<li>Immobilize mice at a prone position on acrylic stage with adhesive tape and apply mineral oil with a cotton swab in the calf and knee (Figure 2A-D).</li>
	<li>Perform a midline incision in the calf from the heel to the knee (Figure 2E,F).</li>
	<li>Dissociate the calf musculature from the skin (Figure 2F,G).</li>
	<li>Expose the popliteal fossa (Figure 2G). Popliteal lymph node will appear as a translucid sphere in the popliteal fossa.</li>
	<li>Remove the popliteal lymph node using microsurgery curved forceps (Figure 2G).</li>
	<li>Alternatively, turn the mouse over on supine position, and approach the popliteal fossa between biceps femoris and semitendinosus for popliteal LN removal by performing a midline incision in the calf from the heel to the knee followed by dissociation of the calf musculature from the skin. 
	NOTE: Popliteal fossa is a shallow depression located at the back of the knee joint. Open carefully to see the popliteal lymph node.</li>
</ol>
5. Lymph node preparation
<ol>
	<li>Place the whole organ in a culture dish with glass bottom (35 mm x 10 mm) and remove the fat that surrounds the organ using microsurgery curved forceps (Figure 1G-I and Figure 2H).</li>
	<li>Centralize the organ in the middle of the dish (Figure 1J and Figure 2H).</li>
	<li>Cover the organ with a fragment of delicate task wipers and keep it soaked with room temperature saline 0.9% or Phosphate Buffer Solution (Figure 1K-M and Figure 2H). 
	NOTE: It is not necessary to wash lymph nodes after removal or to perform the lymph node extraction in the hood.</li>
</ol>
6. Ex-vivo confocal microscopy (Imaging)
<ol>
	<li>Position the Petri dish in the inverted confocal microscope slot (Figure 1N and Figure 2H).</li>
	<li>Image LNs under a confocal microscope (Figure 1O and Figure 2H).</li>
	<li>First, obtain the correct focus by using the conventional light of the confocal microscope with 4x or 10x objective. Then change from the light function to the laser mode. 
	NOTE: If the confocal microscope does not contain a 4x objective, the focus can be perfectly obtained using the 10x objective.</li>
	<li>Adjust the laser power, offset and gain using an isotype-stained, non-stained or non-fluorescent sample (Table 1) to remove autofluorescence and unspecific staining from fluorescent-labeled antibodies such as the ones used in the images showed in this manuscript (BV-421 anti-CD4, BB515 anti-CD19 and PE anti-F4/80).</li>
	<li>Adjust Z and XY positions, using the micrometric and chariot, respectively, on the area of interest in the lymph node.</li>
	<li>Acquire images under 4x, 10x and 20x objectives focusing on the LN structure and cellular distribution. Use 1024 x 1024-pixel definition. 
	NOTE: Acquire a minimum of five images in different fields per objective per animal.</li>
	<li>Analyze images using an image software to separate channels, add scale and colors of interest, and to reconstruct 3D view.</li>
</ol>

<ol><li>Willard-Mack, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Normal+structure%2C+function%2C+and+histology+of+lymph+nodes%5BTitle%5D)+AND+%22Toxicologic+Pathology%22%5BJournal%5D)">Normal structure, function, and histology of lymph nodes.</a> Toxicologic Pathology. 34, 409-424 (2006).</li>
<li>Tas, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Visualizing+antibody+affinity+maturation+in+germinal+centers%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Visualizing antibody affinity maturation in germinal centers.</a> Science. 351, 1048-1054 (2016).</li>
<li>David, B. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Combination+of+Mass+Cytometry+and+Imaging+Analysis+Reveals+Origin%2C+Location%2C+and+Functional+Repopulation+of+Liver+Myeloid+Cells+in+Mice%5BTitle%5D)+AND+%22Gastroenterology%22%5BJournal%5D)">Combination of Mass Cytometry and Imaging Analysis Reveals Origin, Location, and Functional Repopulation of Liver Myeloid Cells in Mice.</a> Gastroenterology. 151, 1176-1191 (2016).</li>
<li>Rezende, R. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((gammadelta+T+cells+control+humoral+immune+response+by+inducing+T+follicular+helper+cell+differentiation%5BTitle%5D)+AND+%22Nature+Communications%22%5BJournal%5D)">gammadelta T cells control humoral immune response by inducing T follicular helper cell differentiation.</a> Nature Communications. 9, 3151(2018).</li>
<li>Nakagaki, B. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Generation+of+a+triple-fluorescent+mouse+strain+allows+a+dynamic+and+spatial+visualization+of+different+liver+phagocytes+in+vivo%5BTitle%5D)+AND+%22Anais+da+Academia+Brasileira+de+Ciencias%22%5BJournal%5D)">Generation of a triple-fluorescent mouse strain allows a dynamic and spatial visualization of different liver phagocytes in vivo.</a> Anais da Academia Brasileira de Ciencias. 91 (suppl 1), e20170317(2019).</li>
<li>Ajami, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Single-cell+mass+cytometry+reveals+distinct+populations+of+brain+myeloid+cells+in+mouse+neuroinflammation+and+neurodegeneration+models%5BTitle%5D)+AND+%22Nature+Neuroscience%22%5BJournal%5D)">Single-cell mass cytometry reveals distinct populations of brain myeloid cells in mouse neuroinflammation and neurodegeneration models.</a> Nature Neuroscience. 21, 541-551 (2018).</li>
<li>Becher, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((High-dimensional+analysis+of+the+murine+myeloid+cell+system%5BTitle%5D)+AND+%22Nature+Immunology%22%5BJournal%5D)">High-dimensional analysis of the murine myeloid cell system.</a> Nature Immunology. 15, 1181-1189 (2014).</li>
<li>McElroy, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fluorescent+LYVE-1+antibody+to+image+dynamically+lymphatic+trafficking+of+cancer+cells+in+vivo%5BTitle%5D)+AND+%22Journal+of+Surgical+Research%22%5BJournal%5D)">Fluorescent LYVE-1 antibody to image dynamically lymphatic trafficking of cancer cells in vivo.</a> Journal of Surgical Research. 151, 68-73 (2009).</li>
<li>Gerner, M. Y., Casey, K. A., Kastenmuller, W., Germain, R. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dendritic+cell+and+antigen+dispersal+landscapes+regulate+T+cell+immunity%5BTitle%5D)+AND+%22The+Journal+of+Experimental+Medicine%22%5BJournal%5D)">Dendritic cell and antigen dispersal landscapes regulate T cell immunity.</a> The Journal of Experimental Medicine. 214, 3105-3122 (2017).</li>
<li>Hauser, A. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Definition+of+germinal-center+B+cell+migration+in+vivo+reveals+predominant+intrazonal+circulation+patterns%5BTitle%5D)+AND+%22Immunity%22%5BJournal%5D)">Definition of germinal-center B cell migration in vivo reveals predominant intrazonal circulation patterns.</a> Immunity. 26, 655-667 (2007).</li>
<li>Allen, C. D., Okada, T., Tang, H. L., Cyster, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Imaging+of+germinal+center+selection+events+during+affinity+maturation%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Imaging of germinal center selection events during affinity maturation.</a> Science. 315, 528-531 (2007).</li>
<li>Arnon, T. I., Horton, R. M., Grigorova, I. L., Cyster, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Visualization+of+splenic+marginal+zone+B-cell+shuttling+and+follicular+B-cell+egress%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Visualization of splenic marginal zone B-cell shuttling and follicular B-cell egress.</a> Nature. 493, 684-688 (2013).</li>
<li>Sipkins, D. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vivo+imaging+of+specialized+bone+marrow+endothelial+microdomains+for+tumour+engraftment%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">In vivo imaging of specialized bone marrow endothelial microdomains for tumour engraftment.</a> Nature. 435, 969-973 (2005).</li>
<li>Cinamon, G., Zachariah, M. A., Lam, O. M., Foss, F. W. Jr, Cyster, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Follicular+shuttling+of+marginal+zone+B+cells+facilitates+antigen+transport%5BTitle%5D)+AND+%22Nature+Immunology%22%5BJournal%5D)">Follicular shuttling of marginal zone B cells facilitates antigen transport.</a> Nature Immunology. 9, 54-62 (2008).</li>
<li>Pereira, J. P., An, J., Xu, Y., Huang, Y., Cyster, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cannabinoid+receptor+2+mediates+the+retention+of+immature+B+cells+in+bone+marrow+sinusoids%5BTitle%5D)+AND+%22Nature+Immunology%22%5BJournal%5D)">Cannabinoid receptor 2 mediates the retention of immature B cells in bone marrow sinusoids.</a> Nature Immunology. 10, 403-411 (2009).</li>
<li>Nakagaki, B. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Immune+and+metabolic+shifts+during+neonatal+development+reprogram+liver+identity+and+function%5BTitle%5D)+AND+%22Journal+of+Hepatology%22%5BJournal%5D)">Immune and metabolic shifts during neonatal development reprogram liver identity and function.</a> Journal of Hepatology. (6), 1294-1307 (2018).</li>
<li>Wang, H., La Russa, M., Qi, L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((CRISPR%2FCas9+in+Genome+Editing+and+Beyond%5BTitle%5D)+AND+%22Annual+Review+of+Biochemistry%22%5BJournal%5D)">CRISPR/Cas9 in Genome Editing and Beyond.</a> Annual Review of Biochemistry. 85, 227-264 (2016).</li>
<li>Roozendaal, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Conduits+mediate+transport+of+low-molecular-weight+antigen+to+lymph+node+follicles%5BTitle%5D)+AND+%22Immunity%22%5BJournal%5D)">Conduits mediate transport of low-molecular-weight antigen to lymph node follicles.</a> Immunity. 30, 264-276 (2009).</li>
<li>Sarder, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((All-near-infrared+multiphoton+microscopy+interrogates+intact+tissues+at+deeper+imaging+depths+than+conventional+single-+and+two-photon+near-infrared+excitation+microscopes%5BTitle%5D)+AND+%22Journal+of+Biomedical+Optics%22%5BJournal%5D)">All-near-infrared multiphoton microscopy interrogates intact tissues at deeper imaging depths than conventional single- and two-photon near-infrared excitation microscopes.</a> Journal of Biomedical Optics. 18, 106012(2013).</li>
</ol>

The authors have nothing to disclose.

The combination of imaging with other techniques, including molecular biology and high dimensional immunophenotyping has enhanced our ability to investigate immune cells in their native context. In fact, while other approaches may require tissue digestion and cell isolation &#8211; which can lead to loss of tissue integrity - the use of in vivo or ex vivo imaging grants a great advantage in investigating different cell subtypes in a geographical fashion3,16. It i...

Lymph nodes (LNs) are ovoid-shaped organs widely present throughout the body with the crucial function of bridging the innate and adaptive immune responses. LNs filter the lymph in order to identify foreign particles and cancerous cells to mount an immune response against them1. Antigen presenting cells (APCs), T cells and B cells work alongside to generate antigen-specific antibodies (humoral immunity) and cytotoxic lymphocytes (cellular immunity) to eliminate the foreign particles and cancerous cells2. Thus, understanding the dynamics of the immune cells present in the lymphatic system will have important implications for the vaccine development and cancer immunotherapy.
The advent of powerful microscopes - including new confocal and super resolution microscopes - has allowed an extraordinary advancement in understanding how different immune cell populations behave in their native environment3. It is now possible to image several simultaneous cell subtypes using a combination of probes with genetically modified mice that express fluorescent proteins under control of specific targets4,5. In fact, high dimensional techniques, including mass cytometry and multi-parametric flow analysis have been crucial to expanding our knowledge on different immune cell compartmentalization and functionality in the health and disease6,7. However, to prepare samples for these techniques, tissues need digestion and cells are separated from their natural milieu to be analyzed in cell suspensions. To surpass these limitations and allow a better translation in biology, the goal of the protocol proposed here is to apply a straightforward methodology to image ex vivo whole lymph nodes using stock confocal microscopes with the benefit of improved speed, tissue structure preservation, and cell viability compared to the conventional immunofluorescence staining. By using this approach, we were able to show that mice deficient for &#947;&#948; T cells, a subtype of T lymphocyte involved in host early defense against pathogens4, have compromised follicles and T cell zones as compared to wild type mice. These findings allowed us to pursue a study in which we demonstrated that &#947;&#948; T cells play a critical role in the homeostasis of lymphoid organs and humoral immune response4. Furthermore, this protocol provides a physiologic pathway for probes and antibodies to reach the lymph node, as they are administered subcutaneously and dissipate through the tissue lymphatic circulation, building on previous reports that used in situ labeling with antibodies to visualize lymphatic-associated structures8,9, germinal center dynamics10,11,12, and targets readily accessible to blood flow13,14,15.

<table><tbody><tr><td>BV421 anti-CD4</td><td>BD Horizon</td><td>562891</td><td>GK1.5; 0.2 mg mL&lt;sup&gt;-1&lt;/sup&gt;</td></tr><tr><td>BB515 anti-CD19</td><td>BD Horizon</td><td>564509</td><td>1D3; 0.2 mg mL&lt;sup&gt;-1&lt;/sup&gt;</td></tr><tr><td>BB515 Rat IgG2a, &amp;kappa; Isotype Control</td><td>BD Horizon</td><td>564418</td><td>R35-95; 0.2 mg mL&lt;sup&gt;-1&lt;/sup&gt;</td></tr><tr><td>BV421 Mouse IgG2b, K Isotype Control</td><td>BD Horizon</td><td>562603</td><td>R35-38 0.2 mg mL&lt;sup&gt;-1&lt;/sup&gt;</td></tr><tr><td>Cellview culture dish</td><td>Greiner-Bio</td><td>627861</td><td>35x10 mm with glass bottom</td></tr><tr><td>Insulin syringes</td><td>BD Plastipak</td><td>-</td><td>Insulin U-100</td></tr><tr><td>Kimwipes</td><td>Kimtech Science Brand</td><td>7557</td><td>size 21 x 20 cm / 100 sheets per box</td></tr><tr><td>Microsurgery curved forceps</td><td>WEP Surgical Instruments</td><td>custom made</td><td>12.5 cm</td></tr><tr><td>Microsurgery curved scissors</td><td>WEP Surgical Instruments</td><td>custom made</td><td>11.5 cm</td></tr><tr><td>Needle</td><td>BD PrecisionGlide</td><td>-</td><td>30 gauge &amp;times; &amp;frac12; inch</td></tr><tr><td>Nikon Eclipse Te + A1R confocal head</td><td>Nikon</td><td>-</td><td>loaded with main 4 laser lines (405, 488, 543 and 647 nm)</td></tr><tr><td>PE anti-F4/80</td><td>BD Pharmigen</td><td>565410</td><td>T45-2342; 0.2 mg mL&lt;sup&gt;-1&lt;/sup&gt;</td></tr><tr><td>PE Rat IgG2a, &amp;kappa; Isotype Control</td><td>BD Pharmigen</td><td>553930</td><td>R35-95; 0.2 mg mL&lt;sup&gt;-1&lt;/sup&gt;</td></tr><tr><td>Zeiss LSM 710 confocal microscope</td><td>Zeiss</td><td>-</td><td>loaded with main 4 laser lines (405, 488, 543 and 647 nm)</td></tr></tbody></table>

visualizing lymph node structure and cellular localization using ex-vivo confocal microscopy

Visualizing Lymph Node Structure and Cellular Localization using Ex-Vivo Confocal Microscopy

Lymph nodes (LNs) are organs spread within the body, where the innate immune responses can connect with the adaptive immunity. In fact, LNs are strategically interposed in the path of the lymphatic vessels, allowing intimate contact of tissue antigens with all resident immune cells in the LN. Thus, understanding the cellular composition, distribution, location and interaction using ex vivo whole LN imaging will add to the knowledge on how the body coordinates local and systemic immune responses. This protocol shows an ex vivo imaging strategy following an in vivo administration of fluorescent-labeled antibodies that allows a very reproducible and easy-to-perform methodology by using conventional confocal microscopes and stock reagents. Through subcutaneous injection of antibodies, it is possible to label different cell populations in draining LNs without affecting tissue structures that can be potentially damaged by a conventional immunofluorescence microscopy technique.

This manuscript shows techniques to remove inguinal and popliteal lymph nodes without damaging their structure following the injection of fluorescent-labeled antibodies to stain specific cell populations in these organs (Figure 1 and Figure 2).
The powerful combination of immunolabeling of LN cells with BV421 anti-CD4 and BB515 anti-CD19 and confocal imaging analysis defined the localization of T cells (CD4+) and B cells (CD19+) in in...

This protocol describes a technique to image different cell populations in draining lymph nodes without alterations in the organ structure.

Lymph nodes (LNs) are organs spread within the body, where the innate immune responses can connect with the adaptive immunity. In fact, LNs are ...

visualizing-lymph-node-structure-cellular-localization-using-ex-vivo

Research

JoVE Journal

Biology

Two-photon imaging has uncovered lymphocyte motility and cellular interactions within the lymph node under basal conditions and durring an immune response 1.  Here, we demonstrate adoptive transfer of  T cells, isolation of lymph nodes, and imaging motility of CD4+ T cells in the explanted lymph node.

Dissection and 2-Photon Imaging of Peripheral Lymph Nodes in Mice

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.

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

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.

Intravital Imaging of the Mouse Popliteal Lymph Node

Recently developed imaging techniques using near-infrared fluorescence (NIRF) may help elucidate the role the lymphatic system plays in cancer metastasis, immune response, wound repair, and other lymphatic-associated diseases.

Non-invasive Optical Imaging of the Lymphatic Vasculature of a Mouse

This video demonstrates a dissection technique to harvest popliteal draining lymph nodes from the mouse hind legs. Lymph nodes play a critical role in bridging innate and adaptive immunity, and harvesting intact lymph nodes — with a preserved tissue structure and cellular viability — can help study local and systemic immune responses.

A Dissection Method to Harvest Murine Popliteal Draining Lymph Nodes for Ex Vivo Imaging

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

Highly Resolved Intravital Striped-illumination Microscopy of Germinal Centers

The extracellular matrix undergoes substantial remodeling during wound healing, inflammation and tumorigenesis. We present a novel intravital immunofluorescence microscopy approach to visualize the dynamics of fibrillar as well as mesh-like matrix components with high spatial and temporal resolution using epifluorescence or two-photon microscopy.

Long-term Intravital Immunofluorescence Imaging of Tissue Matrix Components with Epifluorescence and Two-photon Microscopy

This protocol describes a method to image resident tumor-infiltrating CD8 T cells labeled with fluorescently coupled antibodies within human lung tumor slices. This technique permits real-time analyses of CD8 T cell migration using confocal microscopy.

Ex Vivo Imaging of Resident CD8 T Lymphocytes in Human Lung Tumor Slices Using Confocal Microscopy

A protocol is presented combining tissue clearing with light sheet fluorescence microscopy (LSFM) to obtain three-dimensional and cellular resolution images of the lymphatic vessels and lymph nodes (LNs) collecting the cerebrospinal fluid (CSF) and spinal epidural fluid.

Three-Dimensional Imaging of the Vertebral Lymphatic Vasculature and Drainage using iDISCO+ and Light Sheet Fluorescence Microscopy

Here, a protocol is presented to assess mouse meningeal lymphatic vessels (MLVs), which play an important role in multiple neurologic diseases. The goal of this protocol is to provide an integrated method that enables both functional evaluation of tracer drainage and detailed morphological characterization of the dorsal MLVs network.

Integrated Protocol for Assessing Meningeal Lymphatics: Cisterna Magna Injection, Lymph Node Imaging, Meningeal Dissection, and Whole-Mount Staining

Learn ex-vivo confocal microscopy for visualizing lymph node structure and cellular localization using fluorescent labeled antibodies in mouse models. This method preserves tissue integrity while enabling specific visualization of T and B cell distribution.

Visualizing Lymph Node Structure and Cellular Localization using Ex-Vivo Confocal Microscopy

Explore More Videos