Enzymes 139 



throwing it into the cold fixative; many shrinkage artifacts 

 can be avoided in this way. Acetone is especially recom- 

 mended for acid phosphatase. For other enzymes, cold ethyl 

 alcohol ( 90-100 per cent ) is equally satisfactory and actually 

 preferable because it gives a better cytological fixation and 

 the tissue is easier to handle. A compromise fixative consist- 

 ing of equal volumes of acetone and alcohol will give excel- 

 lent results in the overwhelming majority of cases. Methyl al- 

 cohol is entirely unsuitable as a fixative, since it destroys 

 most enzymes. Most hydrolytic enzymes are reasonably re- 

 sistant to formalin^ and can be fixed in 10 per cent formalin^ 

 (preferably adjusted to pH 6-6.5 with a small amount of 

 phosphate buffer). Again, fixation in the icebox is recom- 

 mended. Formalin-fixed tissues should be cut frozen; embed- 

 ding usually gives very poor results, even if the formalin is 

 washed out carefully. Exceptions will be mentioned under 

 the individual enzymes. 



Dehydration and embedding.— The successful use of frozen 

 sections has been reported repeatedly, especially after for- 

 malin fixation. Alcohol- and, even more, acetone-fixed tissues 

 cut poorly by the freezing method. In addition, the enzymes 

 are not always irreversibly precipitated by these fixatives. In 

 general, embedding is preferable whenever feasible. 



For dehydration, alcohol or acetone can be used. For best 

 results, dehydration should be carried out at icebox tempera- 

 ture. After complete dehydration the pieces can be em- 

 bedded though benzene or chloroform into paraflBn. Regular 

 celloidin-embedding, although it may yield good results in 

 some cases, is not recommended because it requires too long 

 an exposure to alcohol-ether; arid, in addition, the dilute al- 

 cohol in which celloidin blocks are stored is quite harmful to 

 enzymes. Semi-embedding in dilute celloidin has been men- 

 tioned previously (p. 15). 



Acetone-fixed tissues have a tendency to crumble during 



3. Seligman, A. M., Chauncey, H. H., and Nachlas, M. M.: Stain Technol., 

 26:19,1951. 



