Antibody Uptake Assay for Tracking Notch/Delta Endocytosis During the Asymmetric Division of Zebrafish Radial Glia Progenitors

Notch signaling controls sulfate decision and patterning during development in metazoans. We have developed an antibody uptake assay for labeling the notch ligand delta-D and imaging its endocytic trafficking in radial glia progenitors in the developing zebra fish forebrain. The zebrafish hidden brain ventricular injection enables selective Anti-Dld-Atto-647N uptake by myototic radial glia progenitors lining the brain ventricles in development with high efficiency and the long lasting effects.

This protocol can be easily combined with other pharmacological or genetic perturbations utilized at different developmental stages and possibly adapted to the adult brain as well as human pluripotent stem cell derived 2D or 3D brain organoids. After generating the fertilized embryos and incubating them for 18 to 20 hours, remove them from the incubator. Observe them under an epifluorescence microscope using white light first at a 20 times magnification.

Discard dead embryos that appear cloudy or ruptured under the microscope using a glass pipette. Then turn the fluorescent lamp on and choose the RFP filter setting of the microscope. Select the embryos with strong red fluorescence and transfer them to a new dish with egg water.

Now dechorionate the embryos manually with two fine forceps under white light. Hold the chorion with one pair of forceps and make a tear in the chorion with the other forceps. Open the tear carefully using the forceps and make it large enough for the embryo to pass through by gently pushing the embryo with the tips of the other forceps.

Transfer the dechorionated embryos to a new Petri dish with fresh embryonic medium for a quick rinse before microinjection. After pulling fine injection needles on a puller, open the tip of the needle with forceps under a stereo dissection microscope. Make the diameter of the tip around 10 micrometers and the taper angle around 30 degrees.

Now mix 0.5 microliters of the anti-Dld antibody with two microliters of the anti mouse L G Atto-647N antibody by pipetting five to 10 times to conjugate them and incubate at room temperature for at least 30 minutes. At the end of the incubation, add 2.5 microliters of blocking buffer and 0.5 microliters of 0.5%phenol red to the antibody mixture and mixed by pipetting to block any unconjugated antibodies remaining in the mixture. Next, prepare 1%low melting point agarose in the embryo medium.

Heat the agarose-contained mixture at 70 degrees Celsius till the mixture turns transparent. After aliquoting the agarose solution, keep them in a heat block at 40 degrees Celsius. Rinse the embryo in the tube containing 1%low melting point agarose for three seconds.

Place the embryo on an inverted plastic Petri dish lid with individual agarose drops to mount each embryo separately. Lay the embryos flat laterally in the agarose and keep this position until the agarose has solidified at room temperature. Cover all the mounted embryos in the agarose with egg medium.

Put the embedded embryos under the stereo microscope and cover the agarose with egg water. Set the air pressure injector with micro manipulators, 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 micro injector, frontload the prepared glass needle with two microliters of antibody mixture on the micro manipulator. 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 paddle to deliver each injection pulse and tune the injection volume of each delivery to four to five nanoliters.

Then poke the tip of the micro injection needle through the dorsal roof plate of the hind brain, posterior to the rondimere zero-one hinge point and inject about 10 nanoliters of antibody mixture without hitting the brain tissue. Observed 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 micro manipulator.

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 six to eight embryos, peel the agarose with a microsurgical knife to release the embryos from the embedded agarose.

Transfer the micro injected 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 tricane 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 embryo 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 two to three milliliters of 28.5 degrees Celsius preheated embryonic medium containing tricane 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.

The Atto-647N injected embryos showed background fluorescence in the brain ventricle. The anti-Dld-Atto-647N injected zebrafish embryos showed large amounts of internalized fluorescent particles in most cells of the developing forebrain. Time lapse imaging showed the movement of intracellular Anti-Dld-Atto-647N endosomes during cell division.

Most mitotic radial glia progenitors showed anterior or posterior asymmetric segregation of anti-Dld-Atto-647N endosomes into two daughter cells. The asymmetry stabilized toward the end of anaphase, resulting in asymmetric inheritance of notch signaling endosomes by the daughter cells afterward. Make sure the needle has been put into the hidden brand ventricle at the right place and check if there is any leakage after microinjection.

In addition to labeling notch delta signaling, the protocol is applicable for labeling other membrane protein or extracellular proteins using similar strategies, if a good antibody to the extracellular domain is available.