To begin, incubate the fertilized chicken eggs in a humidified egg incubator with the pointed end facing down at 37.5 degrees Celsius. On the sixth day of incubation, or E5, sterilize the eggshell by spraying it with 70%ethanol. Using an egg candler, trace along the perimeter of the airspace above the embryo with a pencil and cover the outlined area with transparent tape.
Gently cut around the traced area with curved scissors without cutting into the embryonic membranes or blood vessels. Discard the top of the eggshell. Add a few drops of saline or cell culture media onto the airspace membrane to wet it so that it will detach easily.
Using fine forceps, carefully pierce the airspace membrane over the top of the embryo. Remove it. Grab the transparent amnion membrane that immediately surrounds the embryo to position the head and inject around 50, 000 cells in five microliters of suitable cell culture media into the optic tectum with a glass micropipette and a Pneumatic PicoPump.
Add a few drops of 50 milligrams per milliliter of ampicillin on top of the embryo and cover the hole in the top of the eggs with clear tape. Place them in a humidified egg incubator until E15 for dissection. The tumors that formed in the E5 optic tectum in vivo after the injection of glioblastoma, or GBM stem cells, or GSCs expressing green fluorescent protein at E15 are shown in this figure.
GSCs attach to the ventricular surface and form invasive tumors in the brain wall. Four colors were used to identify five features in this experiment, green GSCs, white nuclei, white blood vessels, blue integrin alpha-6, and either red SOX2 or red Nestin. These figures show that the GSCs reside in your blood vessels and appear to be migrating along them.
Movies of rotating 3D volume renders to fixed and immunostained slices of in vivo GSC tumors are shown here.
