Visualization of Neural and Vascular Networks in a Chicken Embryo

Begin with a windowed, fertilized chicken egg containing an early-stage embryo with its neural tube excised at the level of somites one to seven.

Take a GFP-labeled donor neural tube from a stage-matched transgenic chicken embryo and implant it into the recipient, ensuring proper anatomical orientation.

Remove excess liquid around the graft to enhance adhesion between the donor and host tissues.

Seal the window with clear tape to prevent dehydration and contamination.

Incubate the egg until the embryo develops to the desired stage.

Remove the tape.

Identify a vitelline vein on the yolk that carries blood toward the embryo and remove the overlying vitelline membrane.

Using a glass needle, steadily inject a lipophilic fluorescent dye into the vein.

Visualize the embryo under a stereofluorescence microscope.

The injected dye binds to the endothelial cells lining the blood vessels, highlighting the vascular network, while the GFP-labeled neural tube enables neural network tracking.

When ready to begin the grafting procedure, carefully transfer the dissected neural tube from the watch glass to the host embryo. Position the neural tube in the correct orientation. And gently push the explant adjacent into the excised region. If necessary, use the micro scalpel to trim the explant to the exact size of the excised region.

Once in the correct position, remove PBS and other fluid to help the donor and host tissues adhere and establish the graft. The successful integration of the grafted GFP positive neural tube into the wild type chicken host can be assessed under a fluorescence microscope. Seal the entire window with 24-millimeter wide clear tape to prevent dehydration and contamination.

Label the chimeric embryo. And return the egg to the incubator for further development.

At the desired experimental time point, retrieve the egg from the incubator. And remove the clear tape to gain access to the embryo. Once visible, locate an accessible vein on the yolk, making sure the blood flow is directed towards the embryo.

Choose a branching point on one of the vitelline veins for the injection location. Use tweezers to remove the vitelline membrane above the chosen injection point by tearing in opposite directions. Next, adjust the diameter of a pulled glass needle to the size of the vein.

Load the needle with 5 to 10 microliters of DiI solution. Swiftly insert the needle into the vein. And blow steadily with the mouth tube to allow the DiI to join the blood flow slowly without forming a clot. The success of the DiI injection can be assessed by briefly viewing the embryo under a fluorescence microscope.