Esterases and Lipases 349 



Buffer working solution: 



stock solution 40.0 ml. 



N NaOH (4%) 20.0 ml. 



dilute to a total of 100.0 ml. 



(Preserve substrate and buffer with \% chloretone or crystal of thy- 

 mol.) 



Calcium chloride: 



calcium chloride 10.0 gm. 



distilled water 100.0 ml. 



Working solution: 



buffer working solution 5.0 ml. 



calcium chloride 2.0 ml. 



substrate 2.0 ml. 



distilled water 40.0 ml. 



If solution becomes turbid, filter through fritted glass or porcelain 

 filter of medium density. 



procedure: 



1. Deparaffinize and transfer slides to absoliue alcohol. 



2. Flood with 0.5% celloidin to protect against loss of enzyme. 



3. Hydrate to water. 



4. Incubate: 12-48 hours. 



5. Wash in distilled water. 



6. Treat with 1% lead nitrate (1 gm./lOO ml. water): 15 mintites 

 (calcium stearate transformed to lead stearate). 



7. Wash in several changes distilled water. 



8. Treat with dilute ammonium sulfide (1:5): few minutes (brown 

 sulfide produced). 



9. Rinse in tap water. 



10. Cotmterstain lightly, hematoxylin and eosin if desired. 



11. Dehydrate. 



12. Clear in crude gasoline or tetra-chloroethylene (avoid xylene, 

 fades stain). 



13. Motnit, using resin in one of above; not xylene. 



results: 



sites — golden brown 



comments: 



Sotirces of error: brownish pigment or calcareotis deposits, pig- 

 ments visible in unincubated section. These may be hemosiderin 



