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

Abstract

Source: Rezende, R. M., et al. Visualizing Lymph Node Structure and Cellular Localization using Ex-Vivo Confocal Microscopy. 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 — with a preserved tissue structure and cellular viability — can help study local and systemic immune responses.

Protocol

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

1. Euthanize mice using CO₂ asphyxiation followed by cervical dislocation.
2. 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).
3. Perform a midline incision in the calf from the heel to the knee (Figure 1E, F).
4. Dissociate the calf musculature from the skin (Figure 1F, G).
5. Expose the popliteal fossa (Figure 1G). The popliteal lymph node will appear as a translucid sphere in the popliteal fossa.
6. Remove the popliteal lymph node using microsurgery curved forceps (Figure 1G).
7. 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.

Results

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), an...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Cellview culture dish	Greiner-Bio	627861	35x10 mm with glass bottom
Kimwipes	Kimtech Science Brand	7557	size 21 x 20 cm / 100 sheets per box
Microsurgery curved forceps	WEP Surgical Instruments	Custom-made	12.5 cm
Microsurgery curved scissors	WEP Surgical Instruments	Custom-made	11.5 cm