Fixative A for Drosophila Embryos

MATERIALS

METHOD

• 1. Prepare a 0.2 m stock of sodium cacodylate buffer and adjust pH to 7.2.

• 2. Prepare a fresh stock solution of 8% paraformaldehyde as follows:

  • i. Add 8 g of paraformaldehyde to 100 mL of H₂O.

  • ii. Place on a hot plate at high setting in a chemical fume hood.

  • iii. Heat with stirring until steaming, but do not boil.

  • iv. Add 5–7 drops (may take a few more or less) of 1 n NaOH, one drop at a time until paraformadehyde goes into solution.

• 3. Open a new vial of 50–70% EM-grade glutaraldehyde.

• 4. In a chemical fume hood, remove acrolein from bottle using a syringe.

  • At a 2% concentration, acrolein is obnoxious, but not harmful. It penetrates tissue rapidly and aides in the ability to rapidly dissect the embryo with minimal overall distortion. If possible, do the dissection in an exhaust hood. At the very least, work in a well-ventilated space where traffic is minimal. It is possible to eliminate acrolein from the fixative, and simply wait longer between puncturing and dissection, because paraformaldehyde also penetrates relatively rapidly.

• 5. Combine appropriate amounts of all components to obtain:

  • 2% paraformaldehyde

  • 2% glutaraldehyde

  • 0.13 m sodium cacodylate

  • 1 mm CaC1₂

  • 2% acrolein

  • 1.5% DMSO

• 6. Always check pH after all components are mixed.

  • The paraformaldehyde stock solution when added will always increase pH variably.