Long-term Orthotopic Transplantation of Tumor Cells into Zebrafish Embryos: An Efficient Method to Deliver Tumor Cells into the Fourth Ventricle of Zebrafish Embryos for Long-term Analysis of Tumor Cell Behavior

Using a transfer pipette, transfer 10 to 20 anesthetized embryos to the periphery of the injection plate. The embryos should fall laterally with the ventricles clearly visible and accessible. Use an angled probe to adjust the embryos as needed and position them away from the outer edge of the injection plate.

Then, use a gel-loading tip to load 1 to 2 microliters of the tumor cell suspension into the injection needle and insert the needle into the manipulator. Next, manually lower the manipulator, holding the needle at a 45-degree angle. Adjust the knobs of the micromanipulator in the x, y, and z directions until the needle is just above and approximately 5 millimeters to the right of the embryo head.

Using a stereomicroscope, slowly adjust the micromanipulator in the x direction until the needle pierces the fourth ventricle of the embryo. Do not allow the needle to pierce the heart or the yolk. For consistent injections, the embryos must be anesthetized. The tumor must be properly dissociated into a cellular suspension and the needle must pierce the fourth ventricle but not extend beyond into the heart.

Push the microinjector foot pedal to inject the tumor cell suspension. When finished injecting, use fresh egg water to gently rinse the injected embryos off the injection plate and into a Petri dish. Now, inspect the injected embryos under a fluorescence stereomicroscope in a dark room. Confirm that the injection pressure, angle, needle size, and cell suspension viscosity result in tumor cells filling 25% to 50% of the ventricle space.

Place the embryos back into the 28 degree Celsius incubator overnight. The next day, assess embryo survival by examining morphological and physiological features, such as normal heart and brain development, as previously described.