a dissection method to harvest murine popliteal draining lymph nodes for ex vivo imaging

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

For popliteal draining lymph node harvest, apply mineral oil to the calf and knee on the injected side of the animal. Next, make a midline incision in the calf from the heel to the knee, and dissociate the calf musculature from the skin.
Expose the popliteal fossa. The popliteal lymph node will appear as a translucent sphere within the fossa. Then, remove the lymph node with the microsurgery curved forceps.

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All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. 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 the acrylic stage with adhesive tape and apply mineral oil with a cotton swab in the calf and knee (Figure 1A-D).</li>
	<li>Perform a midline incision in the calf from the heel to the knee (Figure 1E, F).</li>
	<li>Dissociate the calf musculature from the skin (Figure 1F, G).</li>
	<li>Expose the popliteal fossa (Figure 1G). The 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 1G).</li>
	<li>Alternatively, turn the mouse over on the 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>

<table><tbody><tr><td>Cellview culture dish</td><td>Greiner-Bio</td><td>627861</td><td>35x10 mm with glass bottom</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></tbody></table>

Source: Rezende, R. M., et al. <a href="https://app.jove.com/v/59335">Visualizing Lymph Node Structure and Cellular Localization using Ex-Vivo Confocal Microscopy.</a> J. Vis. Exp. (2019)
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 &#8212; with a preserved tissue structure and cellular viability &#8212; can help study local and systemic immune responses.

<img alt="Figure 1" src="/files/ftp_upload/21578/21578_Figure1.jpg" /> 
Figure 1: 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 the 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 ...

Source: Rezende, R. M., et al. Visualizing Lymph Node Structure and Cellular Localization using Ex-Vivo Confocal Microscopy. J. Vis. Exp. (2019)
This ...

A lymph node is an ovoid-shaped secondary lymphoid organ present throughout the body that functions in bridging innate and adaptive immune responses by allowing interactions between immune cells.
To isolate a murine popliteal draining lymph node &#8212; a node that receives lymph from the hind legs &#8212; begin by taking a euthanized mouse fixed on a stage in a prone position.
Prepare the mouse by applying mineral oil to the calf region of the hind legs. The oil prevents fur deposition around the surgical incision site during skin removal.
Make a midline incision from the heel to the knee in the calf region. Dissociate the skin from the underlying musculature and expose the popliteal fossa &#8212; a shallow depression behind the knee joint.
The popliteal draining lymph node appears as a translucent sphere inside the fossa &#8212; embedded within the adipose tissue of the fossa. Remove the lymph node and place it in a culture dish.
Next, carefully remove the adipose tissue surrounding the node to aid in microscope imaging. Cover the organ with an absorbent sheet soaked in an appropriate buffer. The buffer helps maintain the hydration level to preserve tissue viability.
The popliteal draining lymph node is ready for downstream ex vivo imaging assays.

304 ビュー. ex vivoイメージングのためのマウス膝窩ドレナージリンパ節を採取するための解剖法

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

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