Fixation of Drosophila embryos

General remarks

There are a number of ways to fix tissue and embryos. The method of choice depends on the tissue and the following experiments. For immuno-stainings it is important to obtain a high permeability of the tissue, whereas for EM analysis the number of crosslinks has to be maximised. The strength of fixation depends on the crosslinker (increasing from Methanol, formaldehyd, to glutaraldehyd) and the reaction time. The second important issue is extraction, which always goes along with fixation. Detergents such as tritonX100, saponin or tween-20 may be added to the fixative or used afterwards to extract the tissue. This usually improves the penetrance of the staining.

Collecting embryos

  • collect embryos of the appropriate stage on an apple juice plate
  • add bleach (100%) to plate, swirl to cover all embryos, incubate for at least 1-2 min. Make sure not to incubate for too long in bleach
  • pour the bleach with the embryos through a net, wash plate with water, pour through the net to collect the remaining embryos
  • dip the net with the embryos on a paper to remove remaining liquid, repeat washing and drying

Formaldehyde fixation

  • transfer embryos to a scintillation vial with 5 ml heptan and 4,5 ml PBS 
  • add 0.5 ml formaldehyde (37%)
  • fix for 20 min constantly shaking
  • remove lower layer thoroughly, add 5 ml of methane
  • shake vigorously for 30 sec
  • poped embryos sink, embryos in vitelline membrane stay at interface
  • transfer embryos to an eppendorf tube
  • wash twice with methanol
  • store at -20C

heat fixation

  • heat in a scintillation vial 3 ml of salt solution in microwave, 10 sec
  • add net with embryos to boiling solution, incubate for 10 sec
  • add ice pieces, fill vial with salt solution, put on ice, remove net
  • embryos sink down, decant salt solution
  • add 5 ml heptan, 5 ml methane, shake vigorously
  • save poped embryos 
  • wash with methanol and store at -20C

    • salt solution :0,4% NaCl, 0,03% Triton X-100

fixation for microtubuli staining

Two problems have to be considered when staining for microtubuli: 1. preservation of MT during fixation, 2. permeabilisation
  • fix embryos with 37% formaldehyd for 10 min at room temperature
  • remove vitelline membrane as usual by shaking with methanol and heptan
  • store embryos in methanol
  • incubate embryos in 0.5% TritonX-100 for 2h to permeabilise (saponin is a good alternative to Triton

fixation for EM

  • transfer embryos to a scintillation vial with 5 ml heptan and 5 ml 22,7% glutharaldehyd in 100 mM Na-phosphate pH 7,4
  • shake the vial for 30 min to fix the embryos
  • remove the glutharaldehyd, wash once with Na-phosphate buffer
  • collect embryos in a net, dip on a paper towel
  • transfer embryos with a fine brush to a double sticky tape in a small petri dish, cover with 0,1 M Na-phosphate buffer
  • release embryos from the vitteline membrane with a sharp needle
  • collect embryos

    • Glutharaldehyde, Merck 1.12179.0025, 25% for EM, nach Anderson