Virginie Orgogozo

Embryo antibody staining


 Let flies lay eggs for 4h/ON at 25°C. For D. sechellia, put paper with 10uL of octanoic acid 10% (diluted in EtOH)
 Dechorionate in bleach 2-4min
 Wash with distilled water
 Fix in 4% FA + heptane 20min
 Remove the formaldehyde (bottom phase)
 Add methanol, vortex. All the embryos should sink.
 Wash with methanol 3 times
 Keep at –20°C in methanol if necessary.

Rehydrate in PBT
1) add the same volume of PBT (PBS+Tween), wait until embryos go down
2) remove half of the solution, add the same volume of PBT, wait until embryos go down
3) do 3 complete washes: remove all the liquid, add 1mL of PBT, wait until embryos go down.
4) incubate in PBT for 1h

Coloration
 Primary antibodies ON at 4°C or 1h at RT
 3 washes 20min each in PBT
 Secondary antibodies, Hoechst (Ryan’s tube) dilution 1/1000 1h at RT
 3 washes 10min each in PBT


Mounting
 Remove as much PBT as you can
 Put 55uL of Vectashield mounting medium in the tube and aspirate the embryos as they come up
 put the liquid on a slide
 cover with coverslip