The Histochemical Routine 15 



enzymes. The correct fixative or fixatives will be specified 

 in the description of the individual techniques. 



The fixed tissue is either cut on the freezing microtome or 

 embedded in paraffin or celloidin. Formalin-fixed tissues us- 

 ually give excellent frozen sections, while, after alcohol or 

 especially acetone fixation, frozen sections are very delicate 

 and easily break to pieces. Occasionally, especially when an 

 enzyme is not too sensitive to a relatively short fixation but 

 is sensitive to a long exposure to dehydrating agents and/or 

 heat, a sort of semi-embedding may be carried out as follows: 

 thin slices of the tissue are dehydrated by several changes 

 of absolute alcohol or acetone, a few hours each; subse- 

 quently they are transferred to a mixture of equal parts of 

 alcohol and ether for 2 hours, followed by about 4 per cent 

 celloidin in alcohol-ether (Collodion, U.S. P.) for 12-24 

 hours, hardened in 70 per cent alcohol for a few hours, and 

 finally transferred to water. The entire procedure is prefer- 

 ably carried out at icebox temperature. Tissue blocks infil- 

 trated with thin celloidin by this method can be cut on the 

 freezing microtome; the sections have an excellent con- 

 sistency and no tendency to break up. 



In most cases paraffin or celloidin embedding is possible, 

 and both techniques have their advantages and disad- 

 vantages. Celloidin embedding does not require the appli- 

 cation of heat, and this may be an important advantage when 

 dealing with heat-sensitive enzymes. However, dilute al- 

 cohol, used in the storage of celloidin blocks, is very detri- 

 mental to enz)Tiies, and most of them will be destroyed by it 

 in a short time; therefore, it is imperative to cut and process 

 the blocks promptly. 



Paraffin embedding usually causes considerable inacti- 

 vation of enzymes, although in a completely anhydrous state 

 many proteins will resist the denaturing eflFect of heat re- 

 markably well. However, even traces of water left in the 

 tissue will considerably accelerate the rate of inactivation by 

 heat. That is why thorough dehydration of the tissue is so 



