An Antibody Uptake Assay for Imaging Notch/DeltaD Signaling in Zebrafish Radial Glia Progenitors

Put the embedded embryos under the stereomicroscope and cover the agarose with egg water. Set the air pressure injector with micromanipulators, placing them close to the microscopes. Use the steel gas cylinder containing gaseous nitrogen under high pressure as the air resource.

Once the embryos have been mounted in the agarose, open the gas valve. While using the front-fill module of the microinjector, front-load the prepared glass needle with 2 microliters of antibody mixture on the micromanipulator. Now tune the input pressure to 80 to 90 pounds per square inch and the injection pressure to 20 pounds per square inch.

Calibrate the injection volume using a micrometer under the microscope. Set the tune time duration according to the size of the needle opening from 10 to 120 milliseconds. Tap the pedal to deliver each injection pulse, and tune the injection volume of each delivery to 4 to 5 nanoliters.

Then, poke the tip of the microinjection needle through the dorsal roof plate of the hindbrain, posterior to the rhombomere 0/1 hinge point, and inject about 10 nanoliters of antibody mixture without hitting the brain tissue. Observe the flowing of red fluids in the brain ventricle.

After injection, remove the needle tip from the embryo swiftly by rotating the knob of the micromanipulator. For a successful injection, the red dye of the injected mixture remains in the brain ventricle stably without leaking into the surrounded agarose. Move the mounting plate under the microscope to locate another mounted embryo at a suitable position for repeats.

After injecting 6 to 8 embryos, peel the agarose with a microsurgical knife to release the embryos from the embedded agarose. Transfer the microinjected embryos to a fresh dish with 30 milliliters of embryo medium, and place them at room temperature for the next steps. After 30 minutes, transfer the selected embryos to 10 milliliters of embryo medium.

To mount the embryos, prepare 0.8% low melting point agarose containing the same concentration of tricaine in the tube. Use a glass pipette to immerse the injected embryos in the warm agarose for three seconds. Then immediately remove the embryos from the agarose with the same glass pipette and place the embryos in the center of 35-millimeter glass bottom culture dishes with a drop of agarose from the tube.

Orient the embryos gently with a fiber probe or loading tip to keep the dorsal side of the embryonic brain as close to the glass bottom as possible. Turn the embryo position gently to extend the embryo without curling as the agarose solidifies gradually. Afterward, check the embryoid position by flipping the glass bottom dish over.

Ensure that the whole dorsal forebrain with the correctly mounted embryos can be seen under the microscope. Add 2 to 3 milliliters of 28.5 degrees Celsius preheated embryonic medium containing tricaine to cover the embryo. Place the dish properly on the temperature-controlled stage of the confocal microscope, and adjust the temperature of the imaging chamber to 28.5 degrees Celsius. The embryo is now ready for imaging.