30 STUDIES ON ALKALINE PHOSPHATASE 



they should pass into 50 percent alcohol, then into 25 percent 

 alcohol and distilled water. The section should remain at least 

 5 minutes in the distilled water so that small amounts of pre- 

 formed calcium phosphate may be removed from the section. 



The sections are then incubated in a mixture containing a 

 phosphate ester and calcium. As the enzyme splits off phos- 

 phate from the ester precipitation of calcium phosphate occurs 

 in the sections. When studying tissues of unknown phosphatase 

 concentration, I have found it convenient to incubate trial sec- 

 tions for 2 hours and for 20 hours. With the information pro- 

 vided by these trial sections, it is possible to determine the opti- 

 mal periods for which sections should be incubated to discover 

 the detail of distribution of enzyme in the different sites in the 

 specimen. It is always advisable, when a detailed study is be- 

 ing made after the preliminary trials, to use a series of times of 

 incubation, e.g., for mammalian kidney, 5, 10, 20, 40, and 80 

 minutes. The composition of the incubation mixture which has 

 been used for most of the studies reported here has been: 



Milliliters 



2 percent sodium veronal 20 



2 percent sodium glycerophosphate 20 



2 percent calcium nitrate 10 



2 percent magnesium chloride 2 



Water 48 



100 



In some of the earlier work which is included in this chapter, 

 magnesium was omitted. Magnesium is included to act as an 

 activator of the enzyme. It is not always necessary but will 

 often enhance enzyme activity. 



After incubation, the section should be washed in 2 percent 

 calcium nitrate solution for a few minutes, then taken into 1 

 percent cobalt nitrate solution. This converts the calcium 

 phosphate into cobalt phosphate. The section is then washed 

 in distilled water to remove the excess cobalt nitrate, and then 

 placed in an ammonium sulfide solution, which converts the 

 cobalt phosphate into cobalt sulfide, which is black. The sec- 

 tions may then be mounted in the usual manner. To avoid loss 

 of material from sections due to slight acidity in the washing 

 medium, the calcium nitrate solution, cobalt nitrate solution, 



