210 Microscopic Histochemistry 



substrates in distilled water; keep them in the icebox. The 

 solutions are more or less opalescent and quite viscous. 



2. Prepare a buffered Co acetate solution containing about 

 0.0125 M Co acetate in a 0.025-0.03 M tris-maleate buffer of 

 pH 7.6-7.8. Add about 0.5 ml. of 0.2 M solutions of each of 

 the following salts to 100 ml. of the mixture: MgCL, MnCl2, 

 and CaCh (activators). Add a few crystals of camphor. 



For use, blow 1 ml. of the lauroyl- or myristoylcholine 

 stock solution into 50 ml. of the buffered Co solution. In the 

 case of palmitoylcholine, dissolve 1 ml. of the stock solution 

 in 10-15 ml. of propylene glycol and dilute it with the Co 

 solution to 50 ml. Filter the mixtures. 



Incubate shdes for 6-48 hours ( lauroylcholine, 6-12 hours; 

 myristoylcholine, 12-24 hours; palmitoylcholine, 24-48 

 hours) at 37° C. Wash slides under the tap. Immerse them 

 in a dilute solution of yellow ammonium sulfide made up 

 with 70 per cent alcohol instead of water to hasten the devel- 

 opment of color. Counterstain as desired. Dehydrate and 

 mount. Sites of activity are dark gray to black. With lauroyl- 

 choline the precipitate may be rather coarsely granular. 



B. The thiocholine technique }^^— 



Esters of thiocholine are hydrolyzed by cholinesterases at 

 about the same rates as, or faster than, the corresponding 

 choline analogues. However, the specificity of the acetate 

 for the "true" type of the enzyme is not so strict as that of 

 acetylcholine. ^^^ Both cholinesterases will attack acetylthio- 

 choline; butyryl thiocholine is attacked by the nonspecific 

 type only. The nonspecific enzyme, regardless of the sub- 

 strate used, is almost completely inhibited by 30 minutes' 

 preincubation with 10"® M of diisopropylfluorophosphate 

 (DIPFP), whereas the "true" enzyme is inhibited only to 

 the extent of 40 per cent. Any reaction obtained with bu- 

 tyrylthiocholine is due to the "pseudo"-enzyme only, while 



121. Koelle, G. B., Friedenwald, J. S.: Proc. Soc. Exper. Biol. & Med., 

 70:617, 1949. 



122. Koelle, G. B.: J. Pharmacol & Exper. Therap., 100:158, 1950. 



