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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>

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

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 ...

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

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.

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Research

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Immunology

Immune Systems and Components

Cell Isolation and Culture

1.3K vistas. Fuente: Rezende, R. M., et al. Visualización de la estructura de los ganglios linfáticos y la localización celular mediante microscopía confocal ex-vivo. J. Vis. Exp. (2019) Este video muestra una técnica de disección para recolectar ganglios linfáticos poplíteos drenantes de las patas traseras del ratón. Los ganglios linfáticos desempeñan un papel fundamental en la creación de puentes entre la inmunidad innata y la adaptativa, y la recolección de ganglios linfáticos intactos, con...

Video: Un método de disección para recolectar ganglios linfáticos de drenaje poplíteo murino para obtener imágenes ex vivo - Concepto

Analysis of Lymph Node Volume by Ultra-High-Frequency Ultrasound Imaging in the Braf/Pten Genetically Engineered Mouse Model of Melanoma

Tyr::CreER+,BrafCA/+,Ptenlox/lox&#160;genetically engineered mice (Braf/Pten mice) are widely used as an in vivo model of metastatic melanoma. Once a primary tumor has been induced by tamoxifen treatment, an increase in metastatic burden is observed within 4-6 weeks after induction. This paper shows how Ultra-High-Frequency UltraSound (UHFUS) imaging can be exploited to monitor the increase in metastatic involvement of the inguinal lymph nodes by measuring the increase in their volume.
The UHFUS system is used to scan anesthetized mice with a UHFUS linear probe (22-55 MHz, axial resolution 40 &#181;m). B-mode images from the inguinal lymph nodes (both left and right sides) are acquired in a short-axis view, positioning the animals in dorsal recumbency. Ultrasound records are acquired using a 44 &#181;m step size on a motorized mechanical arm. Afterward, two-dimensional (2D) B-mode acquisitions are imported into the software platform for ultrasound image post-processing, and inguinal lymph nodes are identified and segmented semi-automatically in the acquired cross-sectional 2D images. Finally, a total reconstruction of the three-dimensional (3D) volume is automatically obtained along with the rendering of the lymph node volume, which is also expressed as an absolute measurement.
This non-invasive in vivo technique is very well tolerated and allows the scheduling of multiple imaging sessions on the same experimental animal over 2 weeks. It is, therefore, ideal to assess the impact of pharmacological treatment on metastatic disease.

Melanoma is a very aggressive disease that quickly spreads to other organs. This protocol describes the application of ultra-high-frequency ultrasound imaging, coupled with 3D rendering, to monitor the volume of the inguinal lymph nodes in the Braf/Pten mouse model of metastatic melanoma.

Análisis del volumen de los ganglios linfáticos mediante imágenes de ultrasonido de frecuencia ultra alta en el modelo de melanoma de ratón genéticamente modificado braf/pten

Tyr::CreER+,BrafCA/+,Ptenlox/lox&#160;genetically engineered mice (Braf/Pten mice) are widely used as an in vivo model of metastatic melanoma. Once a ...

Investigación

JoVE Journal

Investigación sobre el cáncer

Sentinel Lymph Node Mapping and Biopsy for Endometrial Cancer at Early Stage with Laparoscopy

Sentinel lymph node (SLN) mapping and biopsy is a promising technique for visualizing and evaluating lymph node status in cancer. This approach has been recommended for low-risk endometrial cancer (EC) patients by authoritative international guidelines, but it has not been performed broadly in China and worldwide. This work aims to describe detailed SLN mapping and biopsy procedures to promote the clinical application. SLN mapping and postoperative pathologic ultrastaging were conducted in a patient with low-risk EC using indocyanine green (ICG) dye to track the SLNs under laparoscopy and resecting them completely for ultrastaging. In conclusion, this protocol describes details of ICG injection, and SLN mapping and biopsy in EC patients based on the experiences gained during clinical practice.

This protocol describes the identification and resection of sentinel lymph nodes to make the operation as easy and minimally invasive as possible.

Mapeo y biopsia de ganglios linfáticos centinelas para el cáncer de endometrio en etapa temprana con laparoscopia

Sentinel lymph node (SLN) mapping and biopsy is a promising technique for visualizing and evaluating lymph node status in cancer. This approach has been ...

Collection and Processing of Lymph Nodes from Large Animals for RNA Analysis: Preparing for Lymph Node Transcriptomic Studies of Large Animal Species

Large animals (both livestock and wildlife) serve as important reservoirs of zoonotic pathogens, including Brucella, Mycobacterium bovis, Salmonella, and E. coli, and are useful for the study of pathogenesis and/or spread of the bacteria in natural hosts. With the key function of lymph nodes in the host immune response, lymph node tissues serve as a potential source of RNA for downstream transcriptomic analyses, in order to assess the temporal changes in gene expression in cells over the course of an infection. This article presents an overview of the process of lymph node collection, tissue sampling, and downstream RNA processing in livestock, using cattle (Bos taurus) as a model, with additional examples provided from the American bison (Bison bison). The protocol includes information about the location, identification, and removal of lymph nodes from multiple key sites in the body. Additionally, a biopsy sampling methodology is presented that allows for a consistency of sampling across multiple animals. Several considerations for sample preservation are discussed, including the generation of RNA suitable for downstream methodologies like RNA-sequencing and RT-PCR. Due to the long delays inherent in large animal vs. mouse time course studies, representative results from bison and bovine lymph node tissues are presented to describe the time course of the degradation in this tissue type, in the context of a review of previous methodological work on RNA degradation in other tissues. Overall, this protocol will be useful to both veterinary researchers beginning transcriptome projects on large animal samples and to molecular biologists interested in learning techniques for in vivo tissue sampling and in vitro processing.

This protocol provides an overview of procedures for the isolation of RNA for the transcriptomic profiling of lymph node tissues from large animals, including steps in the identification and excision of lymph nodes from livestock and wildlife, sampling approaches to provide consistency across multiple animals, and considerations plus representative results for the post-collection preservation and processing for RNA analysis.

Recogida y tratamiento de los ganglios linfáticos de animales grandes para el análisis de ARN: preparación para estudios de nodo de linfa transcriptómicos de grandes especies animales

Large animals (both livestock and wildlife) serve as important reservoirs of zoonotic pathogens, including Brucella, Mycobacterium bovis, Salmonella, and ...

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

Transcripción

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