Alkaline Phosphatase 341 



Alkaline Phosphatase 



Sections containing active phosphatase are incubated in a mixture of 

 calcium salt and phosphate ester, usually sodiuin glycerophosphate, so 

 calcitim phosphate is precipitated at the sites. The enzyme liberates the 

 phosphate ions, which are held on the spot by salts of metals whose 

 phosphates are insoluble. (Magnesium ions are often required as an 

 activator and are added in the form of magnesium sulfate or chloride. 

 The mechanism of activation is unknown.) The insoluble phosphates 

 are made visible either by (1) silver nitrate, producing silver phosphate 

 and metallic silver; or (2) cobalt nitrate and ammonitim sulfide, pro- 

 ducing cobalt phosphate and finally black cobalt sulfide. 



Phosphatase is dissolved or destroyed by ordinary fixatives, but alco- 

 hol or acetone will preserve it. Celloidin or paraffin embedding can be 

 used safely, but paraffin sections must be covered with a film of celloidin 

 to protect against dissolution of enzyme. See Biirstone {I960) for fluores- 

 cent demonstration of alkaline phosphatase. 



fixation: Thin slices (1-2 mm) are fixed at once in chilled acetone, 3 

 changes: 24-48 hotirs. Other fixatives may be 80%, 95%, and abso- 

 lute alcohol, or cold formalin (4°C) followed by freezing method. 



INFILTRATION AND EMBEDDING: For paraffin embedding use chloroform 

 and paraffin at no higher than 58°C melting point, preferably 52°C. 

 Ciu sections 6-10 microns. Do not dry slides at higher than 37 °C. 

 Store all blocks at 6°C until needed. 



solution: 



Incubating Sohuion: 



sodium barbital knife-point full 



(regulates alkalinity, only a bit raises it) 

 3% sodium glycerophosphate (3 gm./lOO ml. 



water) Keep in refrigerator 5-10 ml. 



2% calcium chloride (2 gm./lOO ml. water) ... 20-25 ml. 



1% magnesium sulfate (1 gm./lOO ml. water) . . 10 drops 



distilled water to make 50.0 ml. 



pH should be 9.4; if solution is turbid, filter out free phosphate. 

 procedure: 



1. Deparaffinize (light petroleum can be used), pass throtigh acetone 

 and coat with 0.25-0.5% celloidin; continue hydration. 



