Transmission electron microscopy of thin sections of Drosophila: High-pressure freezing and freeze-substitution

The state of the art in fine-structure preservation for thin sectioning can be achieved by using fastfreezing technology followed by freeze substitution and embedding in resin. Samples prepared by highpressure freezing are estimated to be "fixed" in 20-50 msec. Fast freezing also freezes every cell component regardless of its chemistry. Once frozen, tissues can be processed in a variety of ways before viewing in the electron microscope; here we describe only freeze substitution. In freeze substitution, cells are dehydrated at very low temperatures and cell water is replaced with organic solvent at -80°C to -90°C. At this temperature, large molecules such as proteins are immobilized, yet smaller molecules such as water (ice) can be dissolved and replaced with organic solvents, e.g., acetone. The ideal way to do freeze substitution is with a dedicated freeze-substitution device such as the Leica AFS2 system. These devices allow programming of the times and temperatures needed. Alternatively, if this equipment is not available, freeze substitution can still be performed using items commonly found around the laboratory, as is described here. This protocol is useful for preparing thin sections of Drosophila when the best possible preservation of ultrastructure and antigenicity is required.