Enzymes 205 



The filtered stock solution will keep in the refrigerator for 

 many months. 



Method 



Collodion protection is useful but is permissible only in 

 the case of saturated substrates; the unsaturated ones pene- 

 trate the membrane very poorly. 



To about 45 ml. of 0.05 M tris(hydroxymethyl)-amino- 

 methane or tris-maleate or barbital buffer of pH 7-7.4 add 3 

 ml. of a 2 per cent solution of CaCL, 1 ml. of 5 per cent 

 Tween stock solution, and a few crystals of camphor. Incu- 

 bate slides at 37° C. for 8-24 hours. Rinse shdes in distilled 

 water and immerse them in a 1-2 per cent solution of lead 

 nitrate for 10 minutes at the temperature of the paraffin oven, 

 to transform the Ca soaps into Pb soaps. Rinse slides in re- 

 peated changes of distilled water for 5-10 minutes and im- 

 merse them for about 5 minutes in a dilute solution of light- 

 yellow ammonium sulfide. Wash under the tap, counterstain 

 as desired, dehydrate in alcohols. Clear in gasoline or tetra- 

 chloroethylene; mount in synthetic balsam dissolved in one 

 of the clearing agents mentioned. Xylene will cause rapid 

 fading of the stain. Sites of lipase-esterase activity in a gold- 

 en-brown shade. 



Two minor drawbacks of the method should be mentioned. 

 First, in some cases there is considerable cytolysis in the tis- 

 sue, especially around areas of high activity, making coun- 

 terstaining unsatisfactory. Treatment of the sHde with 10 

 per cent formalin for 10-30 minutes before incubation will 

 help to minimize this complication, without causing any ap- 

 preciable loss in enzymatic activity. Secondly, a certain 

 amount of diffusion of the enzyme (especially in the pan- 

 creas) appears to be imavoidable; specks of loose brown 

 precipitate will almost always be found scattered around 

 sites of high activity. The formalin treatment just mentioned 

 will greatly reduce its amount but wHl rarely succeed in 

 eliminating it completely. Most of the precipitate is formed 



