Enzymes 207 



Method 



Prepare a stock solution of 1 per cent a-naphthyl acetate 

 (Eastman No. 2380) in 50 per cent acetone; it will keep in 

 the refrigerator indefinitely. For use, blow 0.5-1 ml. of the 

 stock solution into about 50 ml. of distilled water, add a few 

 ml. of a 0.2 M solution of disodium phosphate and 20-50 mg. 

 of a diazonium salt (preferably one of the following: Diazo 

 Blue B, Diazo Fast Blue RR, diazotized a-naphthylamine, or 

 Diazo Red RC); stir the mixture and filter it into a Coplin 

 jar. Do not protect sections with collodion because it may be 

 stained by the dye. Incubate slides at a temperature not over 

 20° C. for 5-20 minutes or until the desired intensity of 

 shade is obtained. Rinse in water, counterstain as desired, 

 and mount in glycerol-jelly or balsam, depending on the di- 

 azonium salt used (see p. 170). 



The sensitivity of the a-naphthol method is around 40-50 

 ^M hydrolyzed in 10 minutes per gram of active structure 

 at 10° C. (by Coujard's method). 



2 ) The naphthol AS technique.— The acetate of naphthol 

 AS (the anihde of 2-hydroxy-3-naphthoic acid) is hydro- 

 lyzed about six times slower than the acetate of a-naphthol; 

 this may prove of considerable advantage in the case of 

 highly active tissues, because no precautions are required to 

 prevent diffusion artifacts. On the other hand, it is much less 

 soluble in water than a-naphthol acetate, and, to obtain a 

 high enough concentration, propylene glycol must be added 

 to the incubating mixture. Naphthol AS acetate is not avail- 

 able on the market; it must by synthesized by esterifying 

 naphthol AS with acetic anhydride in pyridine. It forms 

 white crystals, melting at 159° ± 0?5 C. The previously pub- 

 lished^^^ melting point of 161° C. is erroneous. 



Method 



Dissolve 1 per cent of naphthol AS acetate in a mixture of 

 equal parts of acetone and propylene glycol. Keep this stock 



116. Gomori, G.: The histochemistry of esterases. In International re- 

 view of cytology, Vol. 1 ; New York: Academic Press, 1952. 



