336 Histochemistry (chap. 21) 



fore, will cover mainly procedures used to identify enzymatic activity. 



The field is a tremendous and puzzling one of increasingly extensive 

 activity. It is impossible and impractical to include all histochemical 

 methods and their variations in this type of manual. There are avail- 

 able many excellent books in the field. Also there are journals that 

 describe the newest methods and modifications; therefore, only the 

 most familiar (at least to the author) methods are being incorporated in 

 this text. 



For greater detail start with: Cassellman (1959); Danielli (1953); 

 Davenport (I960); Click (1949); Gomori (1952); Gurr (1958); Lillie 

 (1954); and Pearse (I960). 



Freezing-Drying Method for Embedding 



Sometimes enzymes, proteins or other substances can be lost during 

 fixation, dehydration and embedding, and a freezing-drying method 

 must be used. Small pieces (approximately I mm.) are frozen solid 

 instantly with isopentane cooled by liquid nitrogen to a temperature of 

 — I50°C. The tissue must be frozen rapidly to prevent large crystal 

 formation which would disrupt the cells. The initial freezing is 

 commonly called quenching; it stops all chemical reactions that have 

 been going on in the tissues. Immediately after freezing (it must not 

 thaw) the tissue is dehydrated in a drying apparatus in vacuo at a 

 temperature of —30 to — 40°C and the ice is sublimed into water vapor 

 and removed.^ There is no liquid phase present at any time, and there- 

 fore no diffusion of enzymes. 



When the material is dry, it is allowed to rise to room temperature, 

 is infiltrated with degassed paraffin or carbowax and embedded. The 

 advantages of this method are that shrinkage and diffusion artifacts are 

 reduced to a minimum, and enzymes and chemical structures are pre- 

 served. There are some disadvantages; the equipment is bulky and 

 expensive, and only small pieces can be used in order to prevent distor- 

 tion by ice crystals. Because of sensitivity to water, the sections cannot 

 be floated on water, but must be applied directly to warm albiuiienized 

 slides {Mendelow and Hamilton, 1950). Embedded blocks have to be 

 kept stored in a desiccator. 



Carbowax or paraffin can be cut at room temperature, but many 

 enzymes and other tissue constituents necessitate sectioning at low 

 temperatures of —25 to — 20°C or they are lost. Since embedding is 



^ See new "Unitiap," Fisher Scientific Co. 



