184 Microscopic Histochemistry 



terial decomposition of substrates were discussed earlier. 

 Ruyter^^ finds that positive reactions may be obtained after 

 prolonged incubation in alkaline solutions of Ca salts not 

 containing any substrate. The writer was unable to duplicate 

 this observation. 



c) False localizations have been dealt with in detail (pp. 

 142 ff.). 



Method recommended 



Fixation and embedding (see pp. 138 ff. ) . It is advisable, al- 

 though not strictly necessary, to protect slides with collodion. 

 Composition of the incubating mixture: 



3 per cent (±0.1 M) solution of Na glyc- 

 erophosphate 5-10 ml. 



2 per cent (±0.2 M) solution of calcium 



chloride 20-25 ml. 



10 per cent (±0.5 M) solution of magnesi- 

 um chloride (or sulfate) About 10 drops 



Na barbital (powder) 1 knifepointful (0.5-1 g.) 



Distilled water to make 50 ml. 



Instead of barbital, other buffers (p. 221) may be used. 



Should the mixture be turbid ( presence of free phosphate 

 in the substrate), it must be filtered before use. 



Incubate sections around 37° C. for 1-4 hours. 



Wash them under the tap for about 1 minute. 



Immerse in a 1-2 per cent solution of any soluble cobalt 

 salt (acetate, chloride, sulfate, nitrate) for 5 minutes. 



Wash under the tap for 1-2 minutes. 



Immerse in a dilute solution of colorless or light-yellow 

 ammonium sulfide ( a few drops to a Coplin jarful of distilled 

 water) for 5 minutes. 



Wash under the tap. 



Counterstain as desired. Dehydrate and mount. 



Sites of alkaline phosphatase activity appear in black. 



2 ) The azo dye method.— The original form of this method 

 was devised by Menten, Junge, and Green^^ in 1944; modi- 



49. Menten, M. L., Junge, J., and Green, M. H.: J. Biol. Chem., 153:471, 

 1944; Menten, M. L., Junge, J., and Green, M. H.: Proc. Soc. Exper. Biol. & 

 Med., 57:82, 1944. 



