CHAPTER III 

 THE HISTOCHEMICAL ROUTINE 



The first few steps in handling tissues for histochemical in- 

 vestigations are essentially the same as those used in his- 

 tology, except for minor differences due to chemical con- 

 siderations. 



In a few cases fixation is not permissible. Certain sensitive 

 enzymes, especially oxidative, are badly damaged by all 

 known fixatives. In such cases the tissues must be used fresh, 

 in an unfixed condition, either as smears or as frozen sections. 

 Such tissues are not easy to handle; they are extremely frag- 

 ile and are readily cytolyzed by many reagents. Further- 

 more, a number of proteins will remain soluble and diffuse 

 from their original sites unless the proper precautions are 

 taken. The only way to keep proteins insoluble but undena- 

 tured is to use strong ( half-saturated or better ) salt solutions 

 [e.g., (NH4)2S04; NaCl; Na acetate] throughout the histo- 

 chemical procedure, up to the last step, when the final pre- 

 cipitate is produced. In some instances, when the enzyme is 

 resistant to drying out and to moderate heat, the freezing- 

 drying method may be the answer to the problem. 



However, in most cases fixation is possible and preferable. 

 Tissues should be fixed promptly, although a few hours' de- 

 lay, especially if the specimen is refrigerated, seldom causes 

 noticeable changes. 



The choice of the right fixative is important. Fixatives giv- 

 ing good cytological detail and a minimum of artifacts 

 should be preferred whenever possible. Often, however, a 

 compromise is necessary; one may have to sacrifice cytolog- 

 ical excellence to the preservation of the chemical substance 

 investigated. This applies especially to the preservation of 



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