Freezing-Drying Method for Embedding 337 



impractical at these temperatures, tissue is frozen in isopentane 

 (— 160°C) or in dry ice-ethyl alcohol mixttires (— 70°C). The tissue must 

 be maintained at least at dry ice temperature (— 20°C) and sectioned 

 and moimted at this temperature. This is performed inside a cold 

 chamber cryostat^ (~25 to — 20°C). The object holder is precooled and 

 the block frozen to it with a few drops of water. The knife must be 

 sharp and kept as dry as possible. After the section is cut, dip a clean 

 cooled slide in ethyl alcohol or isopentane (— 20°C) and pick up section 

 by touching slide against it (Louis, 1937). A cover glass can be used in 

 the same manner. (Slides sometimes mtist be albumenized.) Dry in a 

 cold room at to — 2°C: 1 hotir with fan. Mellors (1939) thaws them 

 quickly at room temperature for 15 minutes, aided by a warm finger 

 against the undersurface of the slide. If slides must be stored (but only 

 for a short time) place them over desiccant (CaS04) in refrigerator. 



Fitz-William et al. (1960) recommend that immediately before each 

 section is cut the tissue block be coated with 20% polystyrene dissolved 

 in methylene chloride (volatile at cryostat temperature). Wait until a 

 highly reflecting surface, which forms, disappears and then cut section. 

 Curling of sections is minimal and they can be picked up with fine 

 forceps. The polystyrene sokition should be stored in the cryostat. 

 These authors also include complete directions on the care of the 

 microtome used in the cryostat. 



See also Greenhaum (1936) and Coons et al. (1931). 



Ice-Solvent (freeze-substitution) Method 



Freeze-dry methods can be troublesome and costly. A simpler and 

 cheaper method with excellent results is the ice-solvent procedure 

 wherein the tissue is rapidly frozen and then the ice formed ^vithin the 

 tissue is slowly dissolved in a fluid solvent such as ethyl or methyl 

 alcohol. (Other polar solvents also have been tried. See Patten and 

 Brown, 1938.) When the tisstie is free of ice and completely permeated 

 by the cold solvent, it is brought to room temperature and embedded. 

 In addition to being simpler than freeze-drying, other advantages are: 

 no disruptive streaming movements in the tissue — the ice simply dis- 

 solves — and the substitution solution can sometimes be chosen to con- 

 tain a specific precipitant for a specific substance. The method can be 

 used for radioautography, fluorescent preparations, many enzymes, 

 and other cyto-histochemical methods. There can, however, be cases in 

 "which the freeze-drying procedure is preferred — for instance an enzyme 



- Linderstrom-Lang Cryostat, manufactured by Harris Refrigeration Co., Cambridge, 

 Mass.; also, Fisher & Lipshaw advertise Cryostats. 



