Name: visualizing lymph node structure and cellular localization using ex-vivo confocal microscopy
Uploaded: 2019-08-09T13:30:00
Description: Watch this Scientific Journal Video about Visualizing Lymph Node Structure and Cellular Localization using Ex-Vivo Confocal Microscopy at JoVE.com

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 th...

<ol>
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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-1</td>
		</tr>
		<tr>
			<td>BB515 anti-CD19</td>
			<td>BD Horizon</td>
			<td>564509</td>
			<td>1D3; 0.2 mg mL-1</td>
		</tr>
		<tr>
			<td>BB515 Rat IgG2a, &#954; Isotype Control</td>
			<td>BD Horizon</td>
			<td>564418</td>
			<td>R35-95; 0.2 mg mL-1</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-1</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 &#215; &#189; 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-1</td>
		</tr>
		<tr>
			<td>PE Rat IgG2a, &#954; Isotype Control</td>
			<td>BD Pharmigen</td>
			<td>553930</td>
			<td>R35-95; 0.2 mg mL-1</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 in...

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 ...

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