88 MICROSCOPIC TECHNIQUES 



Substrate B. Same as A, but use 3.3 ml. water and 1.7 ml. of 0.1 M 

 sodium fluoride in place of the 5 ml. water. (Substrate B was used 

 to prevent hydrolysis by phosphatase through the action of the 

 fluoride. Actually Allen and Bourne found that it was not needed 

 in their work since no phosphatase action was observed in their 

 particular experiments.) 



Substrate C. Combine 15 ml. of the purified salt soln. (Formula II) 

 with 2.5 ml. of the sodium iodoacetate and 7.5 ml. water. 



PROCEDURE 



1. Fix tissue in 80% alcohol for 24 hr. 



2. Wash in water for 5-10 min. 



3. Prepare frozen sections and place them in either substrate 

 A or C for 1-2 hr. at 37°. (An extraneous dark precipitate forms 

 on the surface of some of the sections with substrate A, but not 

 with C, presumably because of the higher dilution of the substrate 

 in the latter.) 



4. Treat the sections with the cobalt chloride soln. for several 

 hr. and then with the ammonium sulfide soln. for 10 min. 



5. Dehydrate in alcohols, clear in xylol, and mount in balsam. 



6. Prepare control sections to demonstrate preformed phosphate 

 by omitting the treatment with substrate in step 3 and proceeding 

 with steps 4 and 5. 



Result. The formation of a brownish-black precipitate indicates 

 zymohexase activity. 



LIPASE 



Gomori (1945b) adapted the principle of his method for the 

 demonstration of phosphatases in tissue sections to the localization 

 of lipase. The great difficulty encountered in previous attempts has 

 been to find a substrate that is water soluble and whose acid split- 

 product could be precipitated by some ion having no adverse effect 

 on the enzyme. The ordinary esters of mono- and dicarboxylic acids 

 do not meet both of these requirements. Gomori was able to circum- 

 vent the difficulty by the use of some new long-chain fatty acid 

 esters of hexitans and hexides in which most of the hydroxyl groups 

 are etherified. These compounds were developed by Atlas Powder 



