208 Microscopic Histochemistry 



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

 stock solution into 10-15 ml. of propylene glycol. Shake the 

 mixture. Dilute it, under continuous stirring, with water to 

 50 ml. Add a few ml. of 0.2 M phosphate buffer of pH ±: 6.5. 

 The mixture may be slightly opalescent. Add 20-50 mg. of 

 Diazo Garnet GBC Salt; stir, and filter the solution into a 

 Coplin jar. Incubate slides at room temperature for 30 min- 

 utes to 2 hours or until the desired intensity of shade is ob- 

 tained. Rinse sections, counterstain with hematoxylin, dif- 

 ferentiate thoroughly in acid alcohol, wash under the tap, 

 and mount in glycerol-jelly. 



The sensitivity of the naphthol AS method is around 30 

 ^M in 1 hour per gram of active structure, in terms of a-naph- 

 thol (by Coujard's method). 



3) Methods utilizing other naphthol esters.— As men- 

 tioned, /5-naphthyl acetate should not be used if accurate 

 localization is important. However, if, for special reasons, 

 this method is chosen, it can be used exactly as described 

 under the a-naphthol method. The pH of the incubating 

 mixture should be 8-8.3; refrigeration and stirring will be 

 found necessary in most cases. The best diazonium salts to 

 be used with yS-naphthol are Garnet GBC and diazotized a- 

 naphthylamine. The acetate of 6-bromo-yS-naphthol and the 

 benzoates of a-naphthol and naphthol AS are very slow sub- 

 strates, requiring an incubation of several hours; their use is 

 not recommended. 



Barrett and Seligman^^^ suggest indoxyl acetate as a sub- 

 strate; the indoxyl liberated is demonstrated as indigo. An 

 objection to this type of a method has been voiced on page 

 147. 



II. CHOLINESTERASES 



Choline esters used in physiological and pharmacological 

 experiments (acetate, butyrate, benzoate, etc.) are not suit- 

 able for histochemical use, because no reagents are known to 

 precipitate the products of hydrolysis. This difficulty can be 

 circumvented by ( 1 ) using long-chained fatty acid esters of 



117. Barrett, R. J., and Seligman, A. M.: Science, 114:579, 1951. 



