This video demonstrates the different steps in assay for neural induction in chick embryo. First, the host embryo is explanted In new culture, the donor embryo is explanted in saline and henson's node is labeled with fluorescent dye. Henson node is excised from the donor embryo and transplanted to the host embryo.
The host embryo is cultured for 24 hours, fixed and processed for photo oxidation under fey fluorescence. With dab. The embryo is processed for in-situ hybridization with a neural marker.
Hi, I am Delphine from the Labor Fennell at the Center for Environmental and Genomic Medicine at Texas a and M University in Houston. Today we will show you a procedure for performing neural induction assays in the cheek embryo. We use this procedure in the lab to study the effects of antiepileptic drugs on the neural inducing properties of Hansen's node.
So let's get started. For the neural induction assay, eggs need to be prepared. These eggs should be incubated laying on their side in a humid incubator for 13 hours to hamburger and Hamilton stage three plus or HH three plus.
Next, prepare the host embryo using the new culture method, which has been demonstrated previously. In JoVE, cover the surface of the host embryo with 200 microliters saline. This will facilitate later transfer and positioning of the donor graft.
Finally, cover the assembly with an inverted Falcon 35 millimeter culture dish. To explan the donor embryo, open the egg by tapping on the shell with forceps. Remove top of shell and discard.
Remove the thick albumin with forceps and tilt the yolk sack with coarse forceps so that the embryo faces upwards. After removing the albumin, use fine scissors to cut a square of yolk sack around the embryo. Remove the embryo from the yolk with a spoon and place in a dish containing PBS.
Next, use forceps to detach the donor embryo from yolk and area lucita. Once it has been detached, transfer donor embryo to a SIL guard covered dish containing PBS to label and transplant the graft. Use a micro electrode puller to pull 50 microliter glass micro capillary pipettes under the microscope.
Cut the tip of the micro pipette using fine forceps. Place micro pipette in a Petri dish lined with a ribbon of modeling clay. Next, prepare the dye solution, which will be used to label the graft prior to transplantation.
First, prepare a stock of dye eye in one milliliter, absolute ethanol at 0.5%This stock can be stored at negative 20 degrees Celsius in the dark. Then in a heat block set at 45 degrees Celsius mixed pre warmed 10 microliter dai stock to 90 microliters, 0.3 molar sucrose. This temperature is necessary in order to prevent precipitation of dai and formation of insoluble crystals.
Briefly spin the solution on a mini SPECT fuge for five to 10 seconds. The DAI solution is now ready to use using insect pins secured the donor embryo on the bottom of the sill guard covered dish dorsal side up. Once the embryo is secured, backfill the micro pipette with D eye by applying gentle pressure.
Using an aspirator tube assembly. Apply a small bolus of dai to henson's node. Repeat, make sure the entire henson's node is labeled next.
Using a micro capillary pipette or a micro dissecting knife cut henson's node, the dorsal side of the node can be distinguished from the ventral side because it forms a groove, whereas the ventral side of the node is flat. If necessary, mark the dorsal side of the graft with carmine powder. This will ensure that the graft is positioned with proper dorsal ventral orientation.
Using a 10 microliter gilson pipette, transfer the graft to the host embryo using a micro capillary pipette. Bring the graft close to the area lucita area, opaque boundary region at the level of henson's node of host embryo. Next, use a micro capillary pipette or a micro dissecting knife to make a small incision 80 to 100 micrometers in the area.
Lucita area opaque boundary at the level of henson's node of host embryo. Using a micro capillary pipette or a micro dissecting knife. Position the graft so that the ventral side faces towards you and the posterior end is parallel to equivalent region in host embryo O.Once in position remove saline from host embryo, making sure the graft remains positioned in close proximity with host site and process the host embryo for new culture for 18 to 22 hours as described in our previous submission.
After 24 hours of culture, the embryo is removed from the incubator and pinned down in a cigar dish. Containing P-B-S-P-B-S is removed and replaced with 4%para formaldehyde. The embryo is then fixed for 24 hours.
Dispose of the fixative in a suitable hazardous waste receptacle and add PBS to the fixation dish. Remove insect pins with forceps after the pins have been removed. Use a blunt end pasture pipette to transfer the embryo to a scintillation vial containing PBS.
Wash the embryo twice in PBS for five minutes each and once in twist buffer for five minutes after washing, transfer the embryo to a glass cavity slide containing 0.1 milliliters Tris buffer. Replace the tris buffer solution with 0.1 milliliter DAB solution. Dispose of einor tip in bucket containing a bleach solution.
In order to decontaminate DAB, place a cover slip on top of cavity slide under FEI fluorescein isothiocyanate fluorescence focus. The objective of the microscope on the graft donor cells will appear fluorescent red and exposed to FEI fluorescence until all fluorescent red cells have become brown. This happens as a result of the dough fluorochrome being photo converted to insoluble brown crystals by exposure to FEI excitation wavelength.
4 88 nanometers in the presence of dab. This process takes between 20 minutes and two hours. Following completion of the photo oxidation reaction, remove the cover slip using fine forceps.
Transfer embryo to a 20 milliliter scintillation vial containing PBTW or PBS with 0.1%tween 20. Finally deactivate DAB from cover, slip and glass cavity. Slide by incubating an bleach solution to process the embryo for visualization.
Proceed with in-situ hybridization, incubating the embryo for only DIG labeled probe. Next, begin photographing and sectioning as described in Jo Video two. Here is an example of a neural induction assay.
Initially, the graft is positioned in the border between the area lucita and the area alpaca of the host embryo. Both donor and host embryos are stage three plus. After nine hours in culture, the graft starts to differentiate and induce what appears as a miniature stage eight minus head.
After 24 hours in culture, the neural induction is complete following fixation. The embryo is processed for photo oxidation. Initially, donor derived dye eye labeled cells are shown to have proliferated to form a miniature Noor as seen here in red.
After photo oxidation, these cells appear brown. The embryo is then processed with whole mount in situ hybridization for the neural marker. O otx two developed in the laboratory of Dr.Bonelli.
The donor graft is shown to induce the expression of OTX two from host derived tissue. We've just shown you how to perform a neural induction assay in the cheek embryo. When doing this procedure, it's important to remember to remove all fluid in the area surrounding the graft following transplantation using a pipe pet.
This will ensure that the graft is securely positioned and that it'll not move away from the host embryo during the culture. So that's it.Thanks. So watching and good luck with your experiments.
