34 STUDIES ON ALKALINE PHOSPHATASE 



extension of the time of fixation, clearing, infiltration, etc., does 

 not appear to increase the amount of destruction of enzyme. 



In the second series of experiments, large numbers of sections 

 were cut from one block of paraffin-embedded rat kidney. A 

 number of control sections were taken through the whole glyc- 

 erophosphate procedure as quickly as possible, apart from a 

 period of 1 hour for incubation in the glycerophosphate mixture. 

 The experimental group of sections was taken through all steps 

 in the procedure, bar one, as rapidly as the control sections, but 

 it was exposed to this one step in the procedure for 24 hours; 

 i.e., an experimental section was exposed to, e.g., xylol, alcohol, 

 or collodion, or simply kept at 37° in air after flattening, or in 

 distilled water before incubation, or in calcium nitrate, cobalt 

 nitrate, distilled water, ammonium sulfide, or one of the alcohols 

 or xylols after incubation, for 24 hours. The loss of apparent 

 enzyme activity amounted to 100 percent in some of these steps. 

 Further study was made of those steps which caused loss of ap- 

 parent enzyme activity. The results are incorporated in Table 

 II which gives both the duration of each step which has been 

 found to be generally convenient in the laboratory and the 

 maximum duration of the steps which I have found caused no 

 loss of enzyme activity. It is probable that some of the steps 

 may be even more protracted than suggested in the table with- 

 out causing loss of enzyme activity. On the other hand, certain 

 steps, notably k, I, and m, of the table are very likely to cause 

 loss of apparent activity, so that timing of these operations 

 must be very carefully controlled. Although step m (washing 

 in tap water after ammonium sulfide) may cause loss of enzyme 

 activity if protracted, sections may be left for many hours in tap 

 water to which a little ammonium sulfide has been added with- 

 out loss of apparent activity. 



Where small traces of phosphatase activity are being studied, 

 it is advisable to add one drop of 0.1 M disodium phosphate solu- 

 tion to the incubation mixture, and then filter off the precipitate 

 before incubation. This insures that the incubation mixture is 

 saturated in phosphate ion so that any trace of phosphate which 

 is liberated by enzyme activity is more likely to be precipitated. 

 As noted earlier, it is also necessary to add a little sodium 

 veronal to the calcium nitrate, cobalt nitrate, and distilled water 

 which are used after incubation; this is necessary to counteract 



