This procedure begins with isolating the egg from the shell. The yolk sack is cut around the egg and the membrane is peeled and placed over a watch glass. A ring is placed over the embryo.
The vilin membrane is lifted over the ring. The embryo is transferred to a culture dish. The culture dish is transferred to a culture box, which is placed in an incubator, and the embryo is cultured.
Hi, I am Delphine from the Ture Brick Finn at the Center for Environmental and Genomic Medicine at Texas a and m University in Houston. Today we'll show you a procedure for culturing cheek embryos outside of the egg. We use this procedure in the lab to study the effects of antiepileptic drugs on U development in the cheek embryo.
So let's get started. This embryo culturing protocol begins with eggs that have been incubated for 16 hours on their side in a humidified incubator to hamburger and Hamilton. Stage four.
To begin open the egg by tapping on the shell with forceps and removing pieces of the shell all around the shell. Remove the top of the shell and discard at stage HH four. The embryo is almost two millimeters long with a well-defined primitive streak and is surrounded by a pear-shaped zona.Lucita.
Collect the thin albumin by tilting the bottom shell on its side. Then remove the thick albumin with forceps. Place the embryo in a plastic dish inside the Pyrex dish containing panic compton saline.
Finally, remove the remaining albumin with forceps. Now the embryo is ready to be centered on a ring. The next step is to place the embryo on a ring.
To do this first tilt the yolk sack so the embryo faces upwards using fine scissors. Cut the yolk sack at or below the level of the equator. Swiftly peel the vilin membrane with the forceps.
Orient the membrane so that it's granular or nons shiny side faces upwards. Place the membrane on the watch glass. Then place a glass ring on top of the vilin membrane and center the embryo.
Finally, lift the membrane up around the edges of the glass ring. Remove this assembly from the saline dish. Then transfer it to a microscope to finish setting up the culture.
The embryo culture is set up while viewed under a dissecting scope. Using fine forceps, make sure the villin membrane is tightly lifted over the glass ring. Trim the excess villin membrane from the inner edge of the glass ring.
Take care not to pierce the membrane with the pipette or forceps. Next, remove the saline from the outer edge of the ring with a sterile pasture pipette. Gently rinse the embryo with saline solution.
To remove yolk cells and loose membrane material, add 200 microliters of saline to the outer edge of the ring. This will help facilitate the transfer to a culture dish. Finally, cover the assembly with an inverted Falcon 35 millimeter culture dish.
Now the embryo is ready to be cultured. To begin the culture of the embryo, add 2.5 liters of thin albumin to a Falcon 35 millimeter culture dish. Next, add 200 microliters of thin albumin to the inner edge of the lid of the culture dish.
This will seal the culture chamber and prevent dehydration of the embryo using fine forceps. Uncover the embryo by sliding the glass ring along the edge of the watch glass and transfer the assembly to the culture dish. Remove all residual saline from the inner surface of the ring with a Pasteur pipette.
Cover the dish with a lid and place the embryo in a humidified chamber. The embryo can be cultured for up to 24 hours at 38 degrees Celsius. After the embryo has been cultured for 24 hours, it can be fixed in paraform aldehyde.
For other procedures such as in situ hybridization described in the accompanying video, remove the culture from the incubator and immediately place it on ice. Fill the culture dish with ice cold PBS or a desy PBS if the embryos are to be processed for in-situ hybridization. Using forceps, detach the embryo from the vilin membrane.
Using the blunt end of a pasture pipette, transfer the embryo to a fresh culture dish containing ice. Cold PBS or desy PBS. Pin the embryo down using blunt ended forceps and aspirate the PBS from the dish.
Add freshly prepared ice cold. Fixative paraform aldehyde is highly toxic. So these steps should be performed in a chemical hood with protective eyewear and gloves.
The fixation time and temperature will depend on the application to be used for in situ hybridization. Fix overnight at four degrees Celsius for cryosectioning. Fix for six to eight hours at four degrees Celsius for whole Mount Immunohisto chemistry.
Fix for one hour at room temperature after fixing for the appropriate time, remove the fixative solution and properly dispose as chemical waste. Then fill the dish with ice cold PBS or dey PBS. If the embryo will be used for whole mount in situ hybridization or immuno staining, it is desirable to pierce the head and the heart with a needle or a microdissection knife.
This will prevent these structures from trapping probe or antibody in later steps. Now that we've gone through the procedure for fixing embryos, let's have a look at how this culture system can be used to track cell migrations in vitro prior to culture, the embryo is at primitive streak stage. At the end of the culture period, the embryo has developed to HH 10, a length of two to three millimeters, and is visible in the center of the culture dish.
It is possible to label a group of cells with carbo zanine, fluorescent dye eye just before culture and follow their movement throughout culture, period. In this case, cells below henson's node were labeled with Dai. These cells are shown to contribute to progressively developing somites, developing somites.
We've just shown you how to culture chicken roots outside of the egg. When doing this procedure, it's important to remember to work as fast as possible in order to minimize the contact of the embryo with room temperature. So that's it.
Thanks for watching and good luck with your experiments.
