This procedure begins with the collection of embryos on fresh grape juice plates 30 to 60 minutes. After removing the plate from the lake cage, pick up the embryos with a moistened brush and place them on double sticky tape. Gently roll one embryo over the tape using one half of a tweezer until the corion breaks open.
Pick up the embryo and place it gently on the cover. Slip over a layer of het glue. Repeat this until you have enough embryos.
After dehydration, the embryos are ready for injection. Hi, I'm Ingrid Bruce Musher from the laboratory of Dr.Jonathan Sculley in the Department of Molecular and Cellular Biology at the University of California Davis. Today we'll show you a procedure to micro inject drosophilas syncytial embryos.
We use this procedure to study the role of micro ttes and mitral based motors in mitosis. For example, we micro inject antibodies or other inhibitors of specific mitotic proteins. By analyzing the inhibitor effect on mitosis, we are able to determine the target proteins function.
So let's get started. To begin preparations two to three days before the experiment, set up the lay cage by cutting two small holes about one centimeter squared on opposite sides of a plastic bottle. Fill the holes with a cotton piece, allowing air to flow.
Tap the flies into the bottle and then cover it with a grape juice plate. With yeast, keep the leg cage at room temperature flies will lay eggs for up to 10 days at least one day before the experiment. Pull needles for injection and keep them at four degrees Celsius.
On the day of the experiment, prepare and load the needles. First, take the labeled tubulin out of the freezer and put it on ice for five to 15 minutes. Next, dilute it with GPEM buffer and then centrifuge it for 10 to 15 minutes at 13, 000 RPM four degrees Celsius.
Finally, load one to two microliters into the needle and keep it at four degrees Celsius. To prevent temperature induced polymerization in the needle. Before setting up the cover slip, make heptane glue by enrolling double sticky tape and placing it in a 100 milliliter bottle.
Add about 50 milliliters of heptane, seal the bottle and rock it for several days. Before preparing the embryos, make a dehydration chamber that the embryos will be placed in prior to injection. To do this, put one part of a 35 millimeter dish inside of a 100 millimeter Petri dish to make a table add dryer, right, which is anhydrous calcium sulfate around it so that the height of the dryer right is no higher than the table and cover.
To begin preparing the embryos, first, collect them from the lake cage by changing the grape juice plate with yeast every hour, the embryos should be imaged about two hours after the start of the collection. To prepare the cover slip, place it on one side of a microscope, slide and tape the four corners down so it doesn't move. Using a cotton tipped applicator, put one layer of heptane glue in a line on the cover slip.
The glue should not be viscous and should dry in a few seconds. If it is too thick, add more heptane. Finally, put a piece of double sticky tape on the slide next to the cover.
Slip with a moistened brush. Carefully pick up the embryos from the grape juice plate and place them on the double sticky tape on the slide. Next, use one half of a tweezer to roll the embryos over the double sticky tape until the corion breaks open.
Pick up the embryo by gently rolling it over the Corian so it sticks to the tweezer and place it on the heptane glue on the cover. Slip with the long side of the embryo parallel to the long side of the cover slip. Place 10 to 20 embryos in one row.
Remove the cover slip and place it in the dehydration chamber for three to eight minutes. The time depends on the local humidity and the amount to be injected. Next, place the cover slip on a metal chamber with vacuum grease.
Finally, cover the embryos with halo carbon oil to avoid further dehydration. The embryos are now ready for injection. First, find the embryos under a 16 x objective.
Move the embryos away and find the needle without moving the focal plane. Center the needle and move it up without moving it in the X or Y direction. To open the needle, put the edge of the cover slip into the field of view, but not where the needle will be.
And lower the needle to the same focal plane. Very carefully move the cover slip until it hits the needle and gently breaks it open. Move the needle up.
Now bring the embryos into view. Lower the needle into the oil, and to make sure to obtain nice liquid drops from the needle. Steadily move the embryo into the needle, inject a drop into the embryo, and then move the embryo away.
After all the embryos have been injected, they are ready for observation on a confocal microscope with a 60 or 100 x objective. A control embryo should look like the one in this movie. This is an embryo expressing GFP tubulin and RFP histone imaged with a 100 x objective on a spinning disc confocal microscope.
The total time shown is eight minutes. Plots of pole to pole distance are very reproducible and are a reliable measure of success in control embryos. They should look like those shown here for cycles 11 through 13.
Shown in this movie is another control embryo. This one expressing KLP 61 FGFP and injected with r Domine tubulin. This was also imaged with a 100 x objective on a spinning disc confocal, and the total time is seven minutes.
This movie was acquired immediately after Microinjection, and the gradient in the concentration of r Domine tubulin is clearly visible at the start of the movie. These embryos can be used to study the inhibition of Klp 61 F after microinjection of Klp 61 F antibody. We can see the loss of Klp 61 FGFP from the spindles.
We've just shown you how to micro inject drosophilas incisional embryos. When doing this procedure, it is important to remember to perform controls, first practice injecting buffer or rutin alone, and make sure mitosis proceeds normally through cellular. Once all your injections are reliable and not causing damage, you can inject an inhibitor and study the effect of that particular inhibition.
So that's it. Thanks for watching and good luck with your experiments.
