Freeze-dried, plastic-embedded tissue preparation: a review

The freeze-dried, plastic-embedded specimen is a versatile type of animal soft tissue preparation, with attributes that commend it for some types of analytical and morphological studies. The preparation involves rapid freezing, freeze-drying, osmic acid vapor fixation, and embedding in an epoxy resin. This preparation, though difficult and tedious, offers some advantages for quantification and localization of water-soluble constituents in intracellular spaces. When prepared according to established protocol, there is evidence to suggest that the embedded sample faithfully retains intracellular levels of water-soluble constituents that are present at time of cryofixation. The sample can be stored easily and indefinitely and the tissue can be examined in a variety of ways. Thin sections will accept many stains for light and electron microscopy; the sample can be used for some histochemical procedures; and quantification of intracellular electrolytes is possible with electron probe microanalysis using one of several techniques. A major disadvantage of the method is the lack of satisfactory preservation of normal solute distributions in extracellular spaces. Important considerations for instruments equipped with Si(Li) energy detector systems are the decrease in mass fraction of the elements of interest due to embedding material, and complications in analysis for some elements because of the presence of osmium. This latter problem is of little consequence for analysis by wavelength spectrometers; and the increase in sample density, because of the embedding material, can be used to advantage for thick sample analysis.