Drosophila embryo sample preparation

The basic idea is to embed the Drosophila embryos into an Agarose column. For the SPIM registration, we embed fluorescent, sub-resolution beads, too.

Materials need

 * apple juice plates
 * fly cages
 * dry yeast
 * household bleach
 * plastic sieves
 * paint brush
 * low melting point agarose
 * glass capilaries
 * metal plungers
 * fluorescent beads
 * eppendorf tubes
 * glass bottle with blue cap

Equipment needed

 * heat block with shaking
 * vortex
 * microwave

= Embryo collection =

1. Set up a cage with a fly strain expressing fluorescent marker during Drosophila embyronic development. For example Histone-YFP fusion that will mark all nuclei.

2.

To get eggs of a deterministic age, we first starve the flies and then feed them yeast-based food in another cage with fresh agar in the cap:



By switching the flies out of that cage after, say, two hours, the eggs are guaranteed to be at most two hours old.

Now it is time to prepare the Agarose:



Put beads into the agarose and vortex for a good amount of time (one minute at least):



Now prepare the PBS to wash the eggs in:



We will use a sieve like this to filter out the embryos after bleaching off the egg shell:



We use standard bleach:



Now pick the embryos gently with a regular brush:



Put them into the Agarose with the beads:



Now put them into a capillary. You can use the capillary forces to get the embryos into the capillaries. Try to put them as central as possible for the best quality of the reconstruction:



= Further reading =

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