Permeabilization, staining and culture of living Drosophila embryos.

ABSTRACT

The organic solvent octane has been used routinely to permeabilize the hydrophobic vitelline membrane surrounding the Drosophila embryo, thereby allowing the movement of small molecules into the egg. We present evidence that hexane is a more effective permeabilizing agent than octane and compare the effects of these solvents on uniformity of permeabilization and embryonic viability. The ability of each solvent to make the embryo accessible to a range of biological stains was compared. The effect of octane versus hexane permeabilization on subsequent embryonic viability was measured at seven different stages during early embryogenesis. We found that although hexane is a superior solvent for permeabilizing the vitelline membrane, it decreases the viability of embryos exposed between 0 and 3 hr of age. Older embryos treated with either hexane or octane are usually viable. We also showed that molecules with a molecular mass of 984 Daltons or more did not diffuse into the embryo following treatment with either hexane or octane. Results presented here challenge a phase-partition model that has been proposed previously to explain the molecular basis of permeabilization of the Drosophila egg. An alternative model is described as well as an optimized protocol for permeabilizing and staining Drosophila embryos at any stage during early embryogenesis while maintaining viability for subsequent culture.