Drosophila embryo sample preparation

''Note: This protocol is very detailed, suitable for researchers who do not necessarily have experience with handling Drosophila and performing wet-lab experiments. The idea is to have an easily accessible sample (most institutions have at least one Drosophilist or we can send anyone the His-YFP flies) on which one can demonstrate the full functionality of the OpenSPIM system by collecting a long-term time-lapse,

For experts the basic idea is to embed the Drosophila embryos in agarose with sub-resolution fluorescent beads inside a capillary equipped by a plunger. How you get there may differ from what we usually do.

Materials need

 * apple juice plates
 * fly cages
 * dry yeast
 * household bleach
 * plastic sieves
 * paint brush
 * low melting point agarose
 * glass capillaries
 * metal plungers
 * fluorescent beads
 * Eppendorf tubes
 * glass bottle with blue cap, glass beakers
 * metal spatulas
 * white plastic weighing baskets
 * PBST (Phosphate buffered saline) with 0.1% Tween or Triton

Equipment needed

 * heat block with shaking
 * vortex
 * microwave

= Embryo collection =

1. Flies are attracted by the smell of fruit and eat yeast. In order to make them lay a lot of eggs in a way that they can be easily harvested we will use agar plates with apple juice smeared with freshly prepared yeast paste.


 * The apple juice plates should be available in any Drosophila lab. Yeast can be easily prepared by mixing dry bakers yeast with a bit of water in a plastic beaker and mold it with a metal spatula into a thick paste. Don't make the paste too watery otherwise flies will stick to it. Keep the yeast paste in the fridge in a large glass beaker covered with aluminum foil.

2. Next we set up a cage with a fly strain expressing fluorescent marker during Drosophila embryonic development. For example Histone-YFP fusion that will mark all nuclei. The bottom of the cage will be the apple juice plate with yeast paste. Put as many flies as you can find, but they should not be too crowded, i.e. having to crawl on top of each other. It will take some time for the flies to acclimatize to the cage (roughly one day) before they start laying eggs in earnest.


 * Getting the flies in the cage involves anesthetizing them with CO2 and pouring the sleeping flies from the bottles where they usually live in the lab into the cage.

3. In order to get embryos of a specific age, it is necessary to go through the so called pre-lays. Flies tend to hold developing embryos in their oviducts before depositing them. Fresh food stimulates egg laying. Therefore in the morning we will exchange the apple juice plate twice in one hour interval to clear the embryos held by the mothers overnight.


 * The change of the plate depends a little on the design of the cage. The general idea is of course to minimize the escape of the flies and escape they will try. The trick is to invert the cage, tap it quite strongly on the table which will bring most of the flies to the bottom and quickly swapping the plate.

4. The next apple juice plate will contain embryos that we want to image. If the cage is very crowded with healthy young flies (less than one week old), there will be plenty of embryos on the plate after one hour of collection. If the flies are old or sparse, we can let them lay for longer periods. For His-YFP flies it makes sense to start imaging them in SPIM starting at about 2 hours of development. Therefore if we collect from a properly pre-laid productive cage for one hour, in another hour most of the embryos will be at the blastoderm stage of embryogenesis, ready to be imaged. In order to image later stages of development, the plate with embryos needs to be appropriately aged in a 25°C incubator.

5. Drosophila embryos are covered with a non-transparent chorion which has to be removed for live imaging. Dechorionation is done by treating the embryo with 50% solution of house hold bleach for exactly two minutes. The easiest way to do this is to pour 50% bleach directly onto the apple juice plate (it is good to remove any dead flies and the excess of yeast in the middle). Then gently scrape the surface of the plate with a fine brush to loosen the embryos. For the next two minutes occasionally gently shake the plate by hand to move the embryos around in the bleach. Finally after the timer goes of, pour the bleach embryo slurry over the kitchen sink into a plastic sieve. Wash the embryos under cold tap water or by spraying with a plastic squirt until the smell of the bleach goes away (2-3 minutes). Finally rinse the embryos by dipping the plastic sieve several times into three white plastic weighing baskets filled with water. Leave the sieve in the last basket.

6. The embryos should be examined under the stereo-microscope. They will be mostly floating on the surface of the water or stuck to the walls of the sieve. If dechorionation was sufficient embryos will look like smooth prolonged ovoids lacking the characteristic dorsal appendages. Embryos can be left in this state for about half an hour, but they should not dry.

= Further reading =

See also the description of the protocols in the of Stephan Preibisch.