A dye phosphate for the histo- and cytochemical demonstration of alkaline phosphatase, with some observations on the differential behaviour of nuclear and extranuclear enzymes.

t I AHE method most generally employed for the cytochemical localization JL of alkaline phosphatase is that due to. Takamatsu (1939) and Gomori (1939). It depends upon the hydrolysis by the enzyme of jS-glycerophosphate in the presence of calcium nitrate. Calcium phosphate is thus precipitated at the site of enzymic activity, and may be rendered visible by various procedures. The method is unsuitable for tissues containing preformed calcium phosphate, unless they be previously decalcified: furthermore, it involves a number of stages, some of which necessitate a highly critical timing (Danielli, 1946). Lastly, the method as at present employed is not readily adaptable to quantitative histochemical estimations. Another technique, developed mainly by Menten, Junge, and Green (1944), depends upon enzymic hydrolysis of a phenol phosphate, in the presence of a diazonium hydroxide: immediately upon liberation, the free phenol couples with diazonium hydroxide and precipitates in situ as an insoluble dye. This procedure is independent of the phosphate residue, and preformed calcium phosphate does not interfere. Nevertheless, the method involves a number of disadvantages, viz.: