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

This reproducible and easy to perform protocol uses an ex-vivo lymph node imaging strategy to track the draining of in-vivo administered fluorescent labeled antibodies to local secondary lymphoid organs. Demonstrating this technique allows a specific visualization of the procedure, which can facilitate a successful execution of the protocol. This is a very simple and straightforward technique that can be performed by anyone.

The only challenging aspect is the mouse handling, which requires some training. This technique has the benefit of an improved speed tissue exertion preservation and cell viability compared to the conventional fluorescent staining methods. Training the procedure would be Bruna Tatematsu, a technician from the laboratory.

Dilute the antibodies of interest, conjugated to the appropriate fluorophores, in 100 microliters of PBS for inguinal lymph node imaging, or 50 microliters of PBS for popliteal lymph node imaging. For inguinal lymph node imaging, load 100 microliters of the antibody cocktail into a one milliliter insulin syringe, equipped with a 30 gauge by half inch needle, and subcutaneously inject the antibodies into the inner thigh of the experimental mouse. For popliteal lymph node imaging, inject 50 microliters of the antibody cocktail subcutaneously into one paw pad of the experimental animal.

Then return the animal to its home box. For inguinal draining lymph node harvest, after waiting at least three hours from the injection, immobilize the mouse on the acrylic stage with adhesive tape and apply mineral oil to the abdominal skin. Use standard microsurgery scissors and microsurgery curved forceps to make a midline skin incision in the abdomen from the pubis to the xyphoid process, and dissociate the abdominal musculature from the skin.

Make horizontal skin incisions in the top and bottom of the vertical incision line, to create skin flaps on the side of injection, and retract the skin to visualize the lymph node. Secure the skin flap to the acrylic plate, and use the forceps to remove the translucent, usually bilobular spherical inguinal draining lymph node. For popliteal draining lymph node harvest, at least 12 hours after the injection, immobilize the mouse in the prone position on an acrylic stage.

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. To prepare the lymph node for imaging, place the whole organs in a 35 by 10 millimeter culture dish with a glass bottom, and use forceps to remove any fat surrounding the tissue.

Center the organ in the middle of the dish, and cover the tissue with a piece of saline-soaked delicate task wiper. For ex vivo imaging, position the dish in the inverted confocal microscope slot and use the conventional light of the confocal microscope and the 4-10X objective to obtain the correct focus. Change from the light function to the laser mode and use an isotope-stained, non-stained, or non-fluorescent sample to adjust the laser power, offset, and gain to remove the auto fluorescence and any unspecified staining.

Acquire images under the 4, 10 and the 20X objectives focusing on the lymph node structure and cellular distribution and using a 1024 by 1024 pixel definition. Then, use an appropriate imaging analysis software program to separate the channels, to add the scale and colors of interest, and to reconstruct the 3D view. The powerful combination of immunolabeling of lymph node cells with anti-CD4 and anti-CD19 antibodies and confocal imaging analysis allows the localization of T and B cells within the inguinal lymph nodes.

In the popliteal lymph nodes, as in the inguinal tissue, the B cell follicles are surrounded by T cell populations, a hallmark of the lymph node structure. As observed in these images, phagocytes do not internalize the injected antibodies, indicating that the B and T cell staining is specific. Moreover, re-reconstruction of the labeling indicates that the T and B cell staining do not overlap, further confirming the specificity of the staining.

Monocellular cells are observed throughout the inguinal lymph node, including the subcapsular sinus. With the majority of the mononuclear cells expressing CX3XR1, followed by CCR2 and CXCR31CCR2 double positive cells. The same pattern of cell distribution and cell phenotypes is observed in the popliteal lymph node.

Both lymph nodes also exhibit dark regions without mononuclear cell receptor expression that are occupied by lymphocytes. Moreover, mononuclear cells are scarce within the inner area of the lymph nodes, remaining concentrated in the outer areas of the tissue, indicating that these cells primarily occupy the lymph node subcapsular sinus. The antibody mixture needs to be precisely injected into the subcutaneous tissue for the lymphatic system to properly drain the antibodies to the lymph node.

This method can be applied to the biodistribution of fluorescent labeled drugs, and to cell targeting studies of fluorescent labeled nanoparticles.