102 CYTOCHEMISTRY OF PROTEINS 



A typical procedure would be as follows: 



1. A piece of tissue is submitted to freeze-drying and sectioned after 

 embedding in wax. 



2. After removal of the wax and passing through alcohol the sections 

 are placed for 10 minutes in a solution of 0.2 percent tetrazotised benzidine 

 at about 4° C, in sodium veronal buffer. 



3. The sections are then washed for 1 minute in each of three changes 

 of 0.1 TV hydrochloric acid. It is essential that this washing should be 

 very thorough, and the solutions must be changed frequently to secure 

 this. 



4. The sections are then placed for 15 minutes in a suspension of 0.1 

 gram of a- or /3-naphthol suspended in sodium bicarbonate or sodium 

 veronal solution. 



5. The sections are washed in distilled water and mounted in balsam. 



Since the diazonium hydroxides can react with a number of 

 tissue components, it is usually necessary to use blocking agents 

 to prevent the chromogenic reagent from combining with all the 

 groups which are open to attack. Among suitable reagents for 

 blocking purposes are dinitrofluorobenzene, performic acid, and 

 benzoyl chloride. Dinitrofluorobenzene will block all phenols 

 and also histidine ; performic acid destroys tryptophane ; and ben- 

 zoyl chloride destroys histidine, tryptophane, and tyrosine. Some 

 of the possible permutations and combinations of blocking agents 

 are shown in Table VII. 



TABLE VII 



The ability of certain amino acid residues to react with diazonium hy- 

 droxides after treatment with various reagents. 



2 : 4 Dinitro 



Fluoroben- Performic Benzoyl 

 Reagent None zene Acid Chloride 



Histidine -+- ? + — 



Tryptophane + + — — 



Tyrosine + — + — 



The use of diazonium hydroxides for the study of nucleic acids 

 was first suggested by J. S. Mitchell. He suggested that a tissue 

 section should first be subjected to benzoylation and then ex- 

 posed to a diazonium hydroxide. The reactive amino acids would 

 be destroyed by benzoylation, and only the nucleic acids should 

 be free to react. Mitchell's conditions of benzoylation were un- 

 satisfactory. He used benzoyl chloride in sodium hydroxide so- 



