A SIMPLE ETHANOL-BASED FREEZE-SUBSTITUTION TECHNIQUE FOR MARINE INVERTEBRATE EMBRYOS WHICH ALLOWS RETENTION OF ANTIGENICITY

A simple technique has been developed for flash freezing and freeze substituting small (0.5 mm) marine embryos, which effectively preserves both cellular and extracellular components, using inexpensive equipment that is readily available in most laboratories. To achieve this, embryos of the starfish, Pisaster ochraceus, were isolated on copper freeze-fracture EM grids. The embryos were then rapidly frozen by plunging the grids into a supercooled liquid cryogen, and stored in liquid nitrogen. Freeze substitution was carried out by placing the specimens in sealed vials containing anhydrous ethanol at - 90-degrees-C for 4-5 days. Following substitution, the specimens were passively warmed to - 20-degrees-C over 2 h and then to room temperature over a further 2 h. They were then embedded in either JB4 for light microscopy or Epon or LR White resins for transmission electron microscopy. Four different liquid cryogens, Freon 12, ethane, propane, and nitrogen slush, were tested. Freezing in propane, the best cryogen of the four, gave good preservation of the embryonic cells but poor preservation of the extracellular matrix (ECM). To overcome this, the embryos were exposed to four cryoprotective agents, dimethylsulphoxide, glycerol, ethylene glycol and propylene glycol prior to freezing, and the results were assessed. The experiments demonstrated that good preservation of both cells and ECM could be achieved by adding 15% propylene glycol in sea water to the embryos prior to freezing in propane. Material preserved in this manner not only gave excellent morphological results, but the antigenicity of both native antigens of ECM components and antibodies to which the animals had been exposed in vivo were retained. The application of this technique to other tissues and embryos should prove useful in many future studies.