32 STUDIES ON ALKALINE PHOSPHATASE 



color change occurs only in much more strongly alkaline solu- 

 tions. Consequently, unless due care is used, the pH of the 

 diazonium hydroxide solution is not adjusted to pTL 9.2 but to 

 a strongly alkaline value. When the tissue sections are im- 

 mersed in this strongly alkaline solution, the phosphatase is 

 rapidly destroyed and, of course, no cytochemical reaction oc- 

 curs. To avoid this difficulty drops of indicator should be placed 

 in a white tile, and to one of these should be added a drop of 

 the diazonium hydroxide solution. When the diazonium hy- 

 droxide solution is at approximately pH 9.2-9.3, on addition of 

 the drop the mixture will initially turn to the appropriate blue- 

 grey color, which will then rapidly fade. If the solution remains 

 blue, the diazonium hydroxide solution is too alkaline. 



Loss of Enzyme Due to Experimental Techniques 



Under this general heading will be included losses due to diffu- 

 sion of substances out of tissue sections at various stages of the 

 procedure, and also loss of enzyme due to inactivation. 



Effect of Fixation 



The fixative originally recommended by Takamatsu and Go- 

 mori was 80 percent alcohol. The effect of this 80 percent alcohol 

 on phosphatase activity was studied on the test-tube level by 

 the usual biochemical procedure for the study of alkaline phos- 

 phatase. Pieces of rat kidney were ground up in a mortar and 

 the activities of aliquots determined (a) without fixation, (b) 

 after 2 hours' exposure to 80 percent alcohol. No significant dif- 

 ference could be found between the phosphatase activity of the 

 two samples. Further experiments were carried out (c) with 10 ^ 

 frozen sections cut from a block of unfixed rat kidney and 10 fi 

 frozen sections which had been fixed for 2 hours in 80 percent 

 alcohol and 6 hours in absolute alcohol. The sectioned material 

 was ground as before and studied by the biochemical procedure. 

 Again no difference could be found between the enzyme content 

 of the fixed and unfixed material. It may therefore be concluded 

 that no significant loss of alkaline phosphatase occurs as a result 

 of fixation in alcohol. Consequently it was possible to use 

 the deposition of cobalt sulfide in frozen sections from alcohol- 



