SECTION VU. PHOSPHATASES 



AZO-DYE AND NAPHTHOLIC FLUORESCENCE 



TECHNIQUES FOR THE DEMONSTRATION 



OF PHOSPHATASES 



M. S. BURSTONE 



National Institute of Dental Research, National Institutes of Health, 



Public Health Service, Department of Health, Education, 



and Welfare, Maryland, U.S. A. 



Theoretical considérations as well as practical expérience indicate that 

 some form of tissue fixation is usually necessary for the accurate localiza- 

 tion of phosphatases. Frozen-dried, single and double embedded tissues 

 yield highly reproducible results for the démonstration of both acid and 

 alkaline phosphatases. Frozen sections mounted on glass slides (Adam- 

 stone-Taylor cold knife method) also give useful results provided that 

 they are briefly fixed in diacetone alcohol or acétone. 



With référence to simultaneous coupling procédure, naphthol AS- 

 phosphates appear to be the best gênerai purpose substrates. Thèse 

 compounds are rapidly hydrolyzed and couple over a wide pH range with 

 suitable diazonium salts to form highly insoluble, permanent azo-dyes. 

 The introduction of halogen groupings into thèse substrates facilitâtes 

 their use with frozen sections, in that the azo-dye formed is less lipid 

 soluble than that produced from simpler naphthols. 



The new post-coupling procédures are of interest from several stand- 

 points. First, the localizations obtained with them agrée with those 

 obtained with the use of other techniques. Second, certain structural 

 features of the naphthol molécule may be employed in the design of 

 substrates for the démonstration of other hydrolytic enzymes. The na- 

 phtholic fluorescence techniques represent an innovation in phosphatase 

 procédures, in that the released products require no coupling. The method 

 is potentially very sensitive because of the highly fluorescent nature of 

 the enzymatically released product, and may find application in the 

 "in vivo** study of phosphatase activity. 



The possible design of naphtholic substrates for spécifie types of 

 phosphatases is also considered. 



In addition to facilitating more définitive microscopic localizations, 

 thenewer naphtholic substrates shed light upon the nature of substantivity 



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