182 Microscopic Histochemistry 



mixture (0.01-0.02 M, Gomori; 0.03 M, Takamatsu). If this 

 concentration is increased to about 0.1 M, nuclear reaction 

 and other diffusion artifacts are completely eliminated unless 

 incubation is unduly prolonged. Higher concentrations of Ca 

 inhibit phosphatase activity. 



Ruyter^^ proposes the use of Mg in the presence of an am- 

 monia buffer instead of Ca. The precipitate obtained will be 

 magnesium ammonium phosphate. This method is not rec- 

 ommended, because the precipitate is rather coarsely crystal- 

 line and does not permit fine localization. However, the addi- 

 tion of a small amount of Mg (around 0.005 M) to the in- 

 cubating medium is useful because of its activating effect. 



Alkaline phosphatase is not too sensitive to minor varia- 

 tions in temperature; incubation at any temperature between 

 30° and 45° C. will do. 



The length of incubation may be varied between wide 

 limits, depending on the intensity of the reaction desired. 

 However, it should be borne in mind that prolonged incuba- 

 tion, even under optimal conditions, favors diffusion artffacts 

 ( DanieUi,*' Gomori^' ) . It would be difficult to estabhsh a def- 

 inite time limit beyond which it is not safe to go. In the 

 writer's experience, after 4 or 5 hours false localizations be- 

 come quite noticeable, and after 12-16 hours they may be 

 widespread and intense. However, this complication need 

 not arise except in special cases, since prolonging incubation 

 beyond 2-3 hours is seldom, if ever, indicated. 



Although the precipitate is clearly visible in polarized 

 light,'^-^ much sharper pictures are obtained by any of the 

 color reactions mentioned under "Calcium." The best pic- 

 tures are obtained with the use of silver and cobalt salts. 

 Dorfman and Epshtein*^ prefer the Turnbull blue method 



41. Ruyter, J. H. C, and Neumann, H.: Biochim. et biophys. acta, 3:125, 

 1949. 



42. Danielli, J. F.: Nature, 165:762, 1950. 



43. Belanger, L. F.: Proc. Soc. Exper. Biol. & Med., 77:266, 1951. 



44. Dorfman, V. A., and Epshtein, S. M.: Doklady Akad. Nauk S.S.S.R., 

 72:977, 1950. 



