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>

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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 is not surprising that the availability of genetically-modified mouse strains in which cells are specifically targeted to express different fluorophores is rapidly increasing. Importantly, the combination of reporter mice and the injection of the antibody mix is a powerful tool to stain different cell populations in-vivo in the same organ4. Moreover, the popularization of gene editing tools like CRISPR/Cas9 has allowed different groups to customize their mouse strains in a way that virtually any cell type can be now imaged in their bona fide location17. Using this approach, the spatial and functional relationship between different cell types can be assessed in deep details. However, if imaging of the cell movement or dynamic events in vivo are not mandatory, a less complex experimental approach can be used. In this case, antibodies and probes are delivered in vivo, and the organ (or a sample) is directly visualized in the microscope.
Here we described a straightforward protocol that dispensed the use of tissue cryopreservation and cryosectioning that can potentially affect organ structures and allowed the appreciation of immune cells within lymph nodes following in vivo administration of fluorescent-labeled antibodies. These protocols demanded minimal technical and surgical skills and could be adapted to visualize virtually any immune cells in their original location. Importantly, we proposed that antibodies should be administered in a way that they will reach the lymph nodes using the same pathways that antigens and cells travel during an immune response. By injecting fluorescent antibodies in the subcutaneous space, it was possible to mimic all tissues and hemodynamic barriers found in vivo and estimate the chronology of antigen dissipation. In addition to this application, this method could be applied and useful for biodistribution of florescent-labeled drugs and cell-targeting studies of fluorescent-labeled nanoparticles.
However, there are limitations to this method. The amount of antibody required is higher compared to conventional histology methods; however, since conventional immunofluorescence microscopy requires several rounds of tissue preparation and staining in order to optimize the technique and obtain good images, the cost of conventional microscopy can potentially overcome the cost of the high antibody concentration employed in our protocol. Next, based on the known properties of lymphatic antigen drainage into the lymph node9,18, the efficiency of labeling is likely to rapidly decrease with the distance to the lymphatic vasculature due to the size of the labeling reagent employed. Indeed, the black areas in some of the images may result from poor tissue penetration. Only 40-100 &#956;m in depth can be achieved with confocal imaging of LN, and thus only superficial LN regions can be visualized. One way to at least partially overcome this issue is to use fluorophores with better excitation and detection by the microscope. Another alternative approach is to use multiphoton laser microscopy with near-infrared excitation wavelength that has been key to deep tissue imaging and was shown to circumvent severe light scattering issues observed with confocal single photon laser microscopy19. In the case of imaging cells stained by antibodies that were delivered in vivo, only antigens that are expressed on the cell surface can be targeted. If antibodies used have not been validated yet, it becomes critical to perform a corresponding isotype control staining to exclude autofluorescence and validate the labeling. This can be performed in the same animal, i.e., cell targeting antibody mix injected ipsilaterally and isotype control mix injected contralaterally. In addition, the amount of antibody necessary to efficiently stain a cell in vivo may vary between experiments and cell lines and an alternative approach for this limitation is to use genetic-targeted fluorescence expression.
In conclusion, we propose a new protocol for whole lymph node imaging that maintains the tissue architectural integrity, is reproducible, easy-to-perform and use conventional confocal microscopes. This technique demonstrates how simple methods can allow regular laboratory structures to work as poly-functional platforms that enable major advances in immune system investigation.

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

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 inguinal and popliteal LNs. In both organs, B cell follicles were surrounded by T cell populations (Figure 3, Figure 4 and video 1), a hallmark of the LN structure1. To exclude the possibility that phagocytes lining the lymphatic sinuses could capture the injected fluorescent antibodies and result in non-specific labeling of cell markers, PE anti-F4/80 was included in the antibody mix. As shown in Figure 5, phagocytes did not internalize injected antibodies, indicating that B and T cell staining was specific. Moreover, Video 1 shows that T and B cell staining did not overlap, confirming staining specificity.
To investigate the spatial localization of phagocytic mononuclear cells in the LN, inguinal and popliteal LNs from CX3CR1GFP/+ CCR2RFP/+ were imaged. Mononuclear cells were found throughout the inguinal LN, including the subcapsular sinus. The majority of these cells were CX3CR1GFP/+, followed by CCR2RFP/+ and double positive cells (yellow) (Figure 6A-F). The same pattern of cell distribution and cell phenotypes was observed in the popliteal LN (Figure 7A-I). Both inguinal and popliteal LNs showed black regions without CX3CR1GFP/+ CCR2RFP/+, which are occupied by lymphocytes. Moreover, CX3CR1+ and CCR2+ cells were scarce in the inner area of the LNs and concentrated in the outer area, indicating that these cells primarily occupy the LN subcapsular sinus (Figure 6G-I and Figure 7J-L). Thus, the proposed protocol can clearly define major cell populations present in lymph nodes.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59335/59335fig01.jpg" /> 
Figure 1: Inguinal lymph node preparation. 
(A) Subcutaneous injection of FACS antibody master mix into the inner thigh. (B) 3 h after the injection, euthanize the mouse, immobilize the mouse on an acrylic plate with adhesive tape and apply mineral oil to the abdominal skin to prevent fur deposition around the incision. (C) Perform a midline incision in the abdomen from the pubis to the xiphoid process. (D) Dissociate the abdominal musculature from the skin and do a skin-flap. (E) Tape the skin-flap on the acrylic plate. (F) Remove the inguinal lymph node using a microsurgery curved forceps. (G-H) Place the organ in a culture dish (G) and remove the fat that surrounds the organ (H). (I) Illustrative picture showing the organ size after cleaning. J-M) Centralize the organ in the middle of a petri dish (J), cover the organ with a piece of delicate task wipers (K) and keep soaked with warm 0.9% saline or 1x PBS (L, M). (N) Position the Petri dish in the microscope slot. (O) Scan the organ. <a href="https://www.jove.com/files/ftp_upload/59335/59335fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59335/59335fig02.jpg" /> 
Figure 2: Popliteal lymph node preparation. 
(A, B) Subcutaneous injection of FACS antibody master mix into the paw pad. (C, D) 3 h after the injection, euthanize the mouse, immobilize mouse on an acrylic plate with adhesive tape (C) and apply mineral oil to the abdominal skin to prevent fur deposition around the incision (D). (E) Perform a midline incision in the calf from the heel to the knee. (F) Expose the popliteal fossa. (G) Remove the popliteal lymph node using microsurgery-curved forceps. (H) Place the organ in a petri dish and remove the fat that surrounds the organ, centralize the organ in the middle of the Petri dish, cover the organ with a piece of delicate task wipers and keep soaked with warm saline 0.9% or 1x PBS. Position the Petri dish in the microscope slot and scan the organ. <a href="https://www.jove.com/files/ftp_upload/59335/59335fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59335/59335fig03.jpg" /> 
Figure 3: Representative confocal microscopy images of whole inguinal lymph nodes of non-immunized naive mice. 
(A-C) LN B and T cells were stained using CD4 (green; T cells) and CD19 (red; B cells); scale bar = 50 &#956;m. (D-I) Amplification of specific area with 10x objective; scale bar = 30 &#956;m. <a href="https://www.jove.com/files/ftp_upload/59335/59335fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/59335/59335fig04.jpg" /> 
Figure 4: Representative confocal microscopy images of whole popliteal lymph nodes of non-immunized na&#239;ve mice. 
(A-C) LN B and T cells were stained using CD4 (green; T cells) and CD19 (red; B cells); scale bar = 60 &#956;m. <a href="https://www.jove.com/files/ftp_upload/59335/59335fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/59335/59335fig05.jpg" /> 
Figure 5: Representative confocal microscopy images of whole inguinal lymph node of non-immunized naive mice showing phagocytes. 
(A-C) LN B and phagocytic cells were stained using CD19 (green; B cells) and F4/80 (blue; phagocytes); scale bar = 100 &#956;m; (D-F) LN T and phagocytic cells were stained using CD3 (green; T cells) and F4/80 (white; phagocytes); scale bar = 100 &#956;m. <a href="https://www.jove.com/files/ftp_upload/59335/59335fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/59335/59335fig06.jpg" /> 
Figure 6: Confocal microscopy images of whole inguinal lymph nodes of genetically-modified CX3CR1GFP/+ CCR2RFP/+ mice without antibody master mix injection. 
(A-C) LN cell distribution was evaluated with CX3CR1 (green) and CCR2 (red); scale bar = 100 &#956;m. (D-F) Amplification of specific area with 20x objective; scale bar = 50 &#956;m. (G-I) LN cell distribution in the lymph node sinus floor; scale bar = 100 &#956;m. <a href="https://www.jove.com/files/ftp_upload/59335/59335fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/59335/59335fig07.jpg" /> 
Figure 7: Confocal microscopy images of whole popliteal lymph nodes of genetically-modified CX3CR1GFP/+ CCR2RFP/+ mice without antibody master mix injection. 
(A-C) LN cell distribution was evaluated with CX3CR1 (green) and CCR2 (red); scale bar = 200 &#956;m. (D-F) Amplification of entire organ with 10x objective; scale bar = 100 &#956;m. (G-I) Amplification of specific area with 20x objective; scale bar = 50 &#956;m. (J-L) LN cell distribution in the lymph node sinus floor; scale bar = 100 &#956;m. <a href="https://www.jove.com/files/ftp_upload/59335/59335fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Video 1" src="/files/ftp_upload/59335/59335vid01.jpg" /> 
Video 1: The specificity of T and B cell staining&#160;<a href="https://www.jove.com/files/ftp_upload/59335/Video+1.mp4">(Right click to download).</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Laser</td>
			<td>Laser Power</td>
			<td>Gain</td>
			<td>Offset</td>
			<td>Pinhole</td>
		</tr>
		<tr>
			<td>406nm</td>
			<td>20%</td>
			<td>65</td>
			<td>-5</td>
			<td>60&#956;m</td>
		</tr>
		<tr>
			<td>488nm</td>
			<td>25%</td>
			<td>50</td>
			<td>-5</td>
			<td>60&#956;m</td>
		</tr>
		<tr>
			<td>561nm</td>
			<td>25%</td>
			<td>65</td>
			<td>-10</td>
			<td>60&#956;m</td>
		</tr>
	</tbody>
</table>
Table 1: Image acquisition settings.

Watch this Scientific Journal Video about Visualizing Lymph Node Structure and Cellular Localization using Ex-Vivo Confocal Microscopy at JoVE.com

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

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

Nanyang Technological University

Research

JoVE Journal

Biology

13.1K Views.  Brigham and Women's Hospital, Harvard Medical School. This protocol describes a technique to image different cell populations in draining lymph nodes without alterations in the organ structure.

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

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

CD8 T cell are key players in the fight against cancer. In order for CD8 T cells to kill tumor cells they need to enter into the tumor, migrate within the tumor microenvironment and respond adequately to tumor antigens. The recent development of improved imaging approaches, such as 2-photon microscopy, and the use of powerful mouse tumor models have shed light on some of the mechanisms that regulate anti-tumor T cell activities. Whereas such systems have provided valuable insights, they do not always predict human responses. In human, our knowledge in the field mainly comes from a description of fixed tumor samples from human patients, as well as in vitro studies. However, in vitro models lack the complex three-dimensional tumor milieu and, therefore, are incomplete approximations of in vivo T cell activities. Fresh slices made from explanted tissue represent a complex multi-cellular tumor environment that can act as an important link between co-cultured studies and animal models. Originally set up in murine lymph nodes1 and previously described in a JoVE article2, this approach has now been transposed to human tumors to examine the dynamics of both plated3&#160;as well as resident&#160;T cells4.
Here, a protocol for the preparation of human lung tumor slices, immunostaining of resident CD8 T and tumor cells, and tracking of CD8 T lymphocytes within the tumor microenvironment using confocal microscopy is described. This system is uniquely placed to screen for novel immunotherapy agents favoring T cell migration in tumors.

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

CD8 T cell are key players in the fight against cancer. In order for CD8 T cells to kill tumor cells they need to enter into the tumor, migrate within the ...

Cancer Research

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 ...

Immunology and Infection

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

Two-photon imaging has revealed an elegant choreography of motility and cellular interactions within the lymph node under basal conditions and at the initiation of an immune response 1. Here, we present methods for adoptive transfer of labeled T cells, isolation of lymph nodes, and imaging motility of CD4+ T cells in the explanted lymph node as first described in 2002 2. Two-photon imaging of immune cells requires that the cells are fluorescently labeled, either by staining with a cell tracker dye or by expressing a fluorescent protein. We demonstrate the adoptive transfer procedure of injecting cells derived from donor mice into the tail vein of a recipient animal, where they home to lymphoid organs within approximately 15-30 min. We illustrate the isolation of a lymph node and describe methods to ensure proper mounting of the excised lymph node. Other considerations such as proper oxygenation of perfused media, temperature, and laser power are discussed. Finally, we present 3D video images of naive CD4+ T cells exhibiting steady state motility at 37&deg;C.

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.

Two-photon imaging has revealed an elegant choreography of motility and cellular interactions within the lymph node under basal conditions and at the ...

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 ...

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 ...

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

The lymphatic vascular system is an important component of the circulatory system that maintains fluid homeostasis, provides immune surveillance, and mediates fat absorption in the gut. Yet despite its critical function, there is comparatively little understanding of how the lymphatic system adapts to serve these functions in health and disease1. Recently, we have demonstrated the ability to dynamically image lymphatic architecture and lymph "pumping" action in normal human subjects as well as in persons suffering lymphatic dysfunction using trace administration of a near-infrared fluorescent (NIRF) dye and a custom, Gen III-intensified imaging system2-4. NIRF imaging showed dramatic changes in lymphatic architecture and function with human disease. It remains unclear how these changes occur and new animal models are being developed to elucidate their genetic and molecular basis. In this protocol, we present NIRF lymphatic, small animal imaging5,6 using indocyanine green (ICG), a dye that has been used for 50 years in humans7, and a NIRF dye-labeled cyclic albumin binding domain (cABD-IRDye800) peptide that preferentially binds mouse and human albumin8. Approximately 5.5 times brighter than ICG, cABD-IRDye800 has a similar lymphatic clearance profile and can be injected in smaller doses than ICG to achieve sufficient NIRF signals for imaging8. Because both cABD-IRDye800 and ICG bind to albumin in the interstitial space8, they both may depict active protein transport into and within the lymphatics. Intradermal (ID) injections (5-50 &mu;l) of ICG (645 &mu;M) or cABD-IRDye800 (200 &mu;M) in saline are administered to the dorsal aspect of each hind paw and/or the left and right side of the base of the tail of an isoflurane-anesthetized mouse. The resulting dye concentration in the animal is 83-1,250 &mu;g/kg for ICG or 113-1,700 &mu;g/kg for cABD-IRDye800. Immediately following injections, functional lymphatic imaging is conducted for up to 1 hr using a customized, small animal NIRF imaging system. Whole animal spatial resolution can depict fluorescent lymphatic vessels of 100 microns or less, and images of structures up to 3 cm in depth can be acquired9. Images are acquired using V++ software and analyzed using ImageJ or MATLAB software. During analysis, consecutive regions of interest (ROIs) encompassing the entire vessel diameter are drawn along a given lymph vessel. The dimensions for each ROI are kept constant for a given vessel and NIRF intensity is measured for each ROI to quantitatively assess "packets" of lymph moving through vessels.

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.

The lymphatic  vascular system is an important component of the circulatory system that  maintains fluid homeostasis, provides immune surveillance, and ...

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

Besides being a physical scaffold to maintain tissue morphology, the extracellular matrix (ECM) is actively involved in regulating cell and tissue function during development and organ homeostasis. It does so by acting via biochemical, biomechanical, and biophysical signaling pathways, such as through the release of bioactive ECM protein fragments, regulating tissue tension, and providing pathways for cell migration. The extracellular matrix of the tumor microenvironment undergoes substantial remodeling, characterized by the degradation, deposition and organization of fibrillar and non-fibrillar matrix proteins. Stromal stiffening of the tumor microenvironment can promote tumor growth and invasion, and cause remodeling of blood and lymphatic vessels. Live imaging of matrix proteins, however, to this point is limited to fibrillar collagens that can be detected by second harmonic generation using multi-photon microscopy, leaving the majority of matrix components largely invisible. Here we describe procedures for tumor inoculation in the thin dorsal ear skin, immunolabeling of extracellular matrix proteins and intravital imaging of the exposed tissue in live mice using epifluorescence and two-photon microscopy. Our intravital imaging method allows for the direct detection of both fibrillar and non-fibrillar matrix proteins in the context of a growing dermal tumor. We show examples of vessel remodeling caused by local matrix contraction. We also found that fibrillar matrix of the tumor detected with the second harmonic generation is spatially distinct from newly deposited matrix components such as tenascin C. We also showed long-term (12 hours) imaging of T-cell interaction with tumor cells and tumor cells migration along the collagen IV of basement membrane. Taken together, this method uniquely allows for the simultaneous detection of tumor cells, their physical microenvironment and the endogenous tissue immune response over time, which may provide important insights into the mechanisms underlying tumor progression and ultimate success or resistance to therapy.

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.

Besides being a physical scaffold to maintain tissue morphology, the extracellular matrix (ECM) is actively involved in regulating cell and tissue ...

Bioengineering

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 ...

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

The lymphatic system associated with the central nervous system (CNS) includes the lymphatic vasculature that spins around the brain, the spinal cord, and its associated LNs. The CNS-associated lymphatic system is involved in the drainage of CSF macromolecules and meningeal immune cells toward CNS-draining LNs, thereby regulating waste clearance and immune surveillance within CNS tissues. Presented is a novel approach to obtain three-dimensional (3D) and cellular resolution images of CNS-associated lymphatics while preserving the integrity of their circuits within surrounding tissues. The iDISCO+ protocol is used to immunolabel lymphatic vessels in decalcified and cleared whole mount preparations of the vertebral column that are subsequently imaged with light sheet fluorescence microscopy (LSFM). The technique reveals the 3D structure of the lymphatic network connecting the meningeal and epidural spaces around the spinal cord to extravertebral lymphatic vessels. Provided are 3D images of the drainage circuits of molecular tracers previously injected into either the CSF via the cisterna magna or the thoracolumbar spinal parenchyma. The iDISCO+/LSFM approach brings unprecedented opportunities to explore the structure and function of the CNS-associated lymphatic system in neurovascular biology, neuroimmunology, brain and vertebral cancer, or vertebral bone and joint biology.

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

The lymphatic system associated with the central nervous system (CNS) includes the lymphatic vasculature that spins around the brain, the spinal cord, and ...

Lymphatic and Blood Network Analysis During Obesity

Lymphatic collecting vessels and lymph nodes are inevitably embedded in adipose tissue. The physiological significance of this observation remains still not elucidated. However, obesity is characterized by impaired lymphatic function and increased vessel permeability. Inversely, lymphatic dysfunction induces obesity in mice, suggesting a significant interplay between lymphatic vessels and the adipose tissue. Therefore, understanding factors leading to lymphatic dysfunction might open new therapeutic windows to prevent obesity and associated comorbidities. The first step in this process requires a precise and detailed visualization of the lymphatic network in healthy and inflamed adipose tissue. Here, we describe a rapid, inexpensive, and efficient method that allows to label and analyze lymphatic and blood vessels. This approach takes advantage of the skin-draining brachial lymph node localization within the subcutaneous adipose tissue. The lymphatic arborization of this tissue can be revealed by injecting fluorochrome-conjugated lectins subcutaneously. Moreover, the in vivo labeling approach provides a way to evaluate lymphatic vessel density and functions. Coupled to blood vessel, adipocyte and immune cell staining, the protocol allows for high-resolution mapping of the subcutaneous adipose tissue by 3D imaging.

Obesity is a growing global public health issue. It has been previously associated with lymphatic dysfunction, suggesting a vital crosstalk between the adipose tissue and the lymphatic system. Here, we propose an accessible methodology allowing the distinct labeling of blood and lymphatic vasculatures within the subcutaneous adipose tissue.

Lymphatic collecting vessels and lymph nodes are inevitably embedded in adipose tissue. The physiological significance of this observation remains still ...

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

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Rozdziały w tym wideo

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

Intravital Microscopy of the Inguinal Lymph Node

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

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

Intravital Imaging of the Mouse Popliteal Lymph Node

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

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

Highly Resolved Intravital Striped-illumination Microscopy of Germinal Centers

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

Lymphatic and Blood Network Analysis During Obesity