78 MICROSCOPIC TECHNIQUES 



ALKALINE PHOSPHATASE* 



The same staining technique for the visualization of alkaline phos- 

 phatase activity was developed independently and simultaneously, 

 oddly enough, by Gomori ( 1939) in Chicago and Takamatsu ( 1939) 

 in Japan. Their method was based on the finding that, when sections 

 of tissue were placed in an alkaline medium containing sodium 

 glycerophosphate, the sites of the enzymatic liberation of phosphate 

 could be determined if calcium ions were present to precipitate the 

 phosphate as it was formed. The deposit of calcium phosphate then 

 could be converted to a more easily visualized black precipitate of 

 cobalt sulfide or metallic silver. Gomori ( 1939) , Hepler et al. ( 1940) , 

 Takamatsu (1939), and Kabat and Furth (1941) have employed 

 the von Kossa silver stain; but, as Bourne (1943) has indicated, it 

 is probably inferior to the cobalt stain used extensively in the latter 

 work of Gomori ( 1941a, 1943) . 



The specificity of the stain for phosphatase has been demonstrated 

 by Gomori (1939, 1941a) and Kabat and Furth (1941), and in a 

 critical study later Danielli (1946b) claimed reliability for the 

 localizations obtained. Preformed insoluble calcium salts will give a 

 positive test and therefore these should either be removed by treat- 

 ing the sections, before incubation with substrate, with citrate buffer 

 of pH 4.5 to 5.0 for 15 min. (Gomori, 1946c), or control sections 

 stained to demonstrate the preformed salts should be compared to 

 the sections treated to visualize the enzyme reaction. Of course the 

 former is preferable. 



Tissues too hard to be sectioned without decalcification present 

 a particular problem since phosphatase is destroyed by the usual 

 processes of decalcification. Kabat and Furth (1941) circumvented 

 this difficulty to some degree by the use of 10% diammonium citrate, 

 which they found could effect certain decalcifications without 

 damaging phosphatase. Bourne (1943) has proposed that small 

 pieces of bone tissue be fixed in 80% alcohol, treated with the sub- 

 strate medium and then with cobalt solution and sulfide to convert 

 the calcium phosphate precipitate to one of cobalt sulfide, and finally 

 subjected to decalcification with trichloroacetic acid. Cobalt sulfide 

 is insoluble in trichloroacetic acid and hence the decalcification can 

 be performed as a final step. Controls can be made by following the 



*See Bibliography Appendix, Ref. 7. 



