Microinjection of Live Drosophila Embryos: Early Delivery of Reagents to the Developing Embryo

This protocol is an excerpt from Brust-Mascher and Scholey, Microinjection Techniques for Studying Mitosis in the Drosophila melanogaster Syncytial Embryo, J. Vis. Exp. (2009).

Recipes:

Grape Juice plates:

• 5.5g bacto agar

• 14.5 g dextrose or glucose

• 7.15 g sucrose

• 45 ml grape juice concentrate (100% juice).

• 204.5 ml H₂0

• 625 μl 10N NaOH

Mix all ingredients and microwave until boiling.
Add 2.8 ml acid mix (acid mix: 20.9 ml propionic acid, 2.1 ml phosphoric acid, 27 ml H₂0)

Mix and pour onto 35 mm Petri dishes. Let solidify at room temperature for one or two days. If plates are not going to be used soon, seal with parafilm and keep at 4 °C (allow to equilibrate to RT before using).

Yeast paste:
Dissolve about one teaspoon of yeast (Sigma YSC2, yeast from Saccharomyces cerevisiae type II) in water to form a thick paste. Place a small amount of this on each grape juice plate just prior to use.

Injection buffer:

• 150 mM K-Aspartate

• 10 mM K-phosphate

• 20 mM imidazole, pH 7.2

Heptane glue:
Unroll double sticky tape and place in a 100ml bottle, add about 50ml heptane, seal the bottle, and rock for several days. When using, add heptane if the glue is too thick.

Dehydration chamber:
Take a 100 mm Petri dish, put one part of a 35 mm dish inside to make a "table" and add Drierite (anhydrous calcium sulfate) around it so that the height of Drierite is no higher than the "table" and cover. The coverslip with embryos will be placed on this "table" for dehydration before injection. It is a good idea to use at least some indicating Drierite and change it when it has changed color.

Protocol:
This protocol can be used for injection of virtually any soluble reagent into the Drosophila syncytial embryo: For example, either a fluorescent protein for observation or a target protein inhibitor or both.

1. Embryo collection

1. Put a new grape juice plate on the lay cage.
2. Remove this plate after one hour, this is the first collection of the day and often is not very good. It can be discarded.
3. Change the plate every hour to keep collecting embryos for one hour each.

2. Coverslip preparation

1. Put a 50 x 22mm coverslip on one side of a microscope slide. Tape the four corners to the slide so it doesn't move.
2. Using a cotton tipped applicator, put one layer of heptane glue in one line on the coverslip (the glue should not be viscous and should dry in a few seconds, otherwise add more heptane).
3. Put a piece of double sticky tape on the slide toward the side of the coverslip.

3. Embryo preparation

NOTE: Embryos should be imaged about 2 hours after start of collection, so begin the following steps allowing sufficient time to finish them within this time-frame.

1. With a moistened brush carefully pick up the embryos from the grape juice plate and place on double sticky tape on slide.
2. Using the outer part of the tweezers, roll the embryos over the double sticky tape until the chorion (the outer membrane) breaks open.
3. Pick up the embryo by gently rolling it over the chorion so it sticks to the tweezer and place it on the heptane glue on the coverslip with the long side of the embryo parallel to the long side of the coverslip. Set up 10 to 20 embryos in one row.
4. Remove the coverslip and place it in the dehydration chamber for 3 to 8 minutes (this depends on the humidity of the room and the amount to be injected).
5. Place on a metal chamber with vacuum grease.
6. Cover the embryos with halocarbon oil 700 to avoid further dehydration. Embryos are now ready for injection.

4. Embryo injection

1. Find the embryos under a 16x objective.
2. Move the embryos away, and find the needle without moving the focal plane.
3. Center the needle and move it up without moving it in x or y direction.
4. If the needle is not open, put the edge of the coverslip in the field of view, but not where the needle will be, and lower the needle to the same focal plane. Very carefully move the coverslip until it hits the needle and gently breaks it open. If the needle is open, go to step 5. (Needles can be opened with hydrofluoric acid before filling).
5. Put the embryos in view (but not where the needle will be), lower the needle into the oil and make sure you obtain nice liquid drops from the needle.
6. Carefully but steadily move the embryo into the needle and inject a drop into the embryo and move the embryo away. (Or follow the instructions of your injection apparatus).
7. After all the embryos have been injected, they are ready for observation on a confocal microscope.