GENERAL STATUS OF TECHNIQUES 49 



Consequently, a series of fixatives were made up as indicated 

 in Table V. Into these fixatives were placed pieces of rat kid- 

 ney approximately 1 millimeter thick. The fixative was al- 

 lowed to act for 2 hours, after which the tissue was washed in 

 water, frozen sections were prepared, and the sections were 

 incubated for logarithmic series of times for each fixative. As 

 will be seen in the table, where the fixation was generally satis- 

 factory from a cytological point of view the enzyme in kidney 

 tissue was restricted to the brush-border region and to the 

 nuclei. This conclusion was reinvestigated later with the freeze- 

 drying method. On the whole, the general nature of the con- 

 clusions reached with chemical fixatives was confirmed. But 

 it was also found that with all the chemical fixatives some dif- 

 fusion of phosphatase into the nuclei appeared to occur. With 

 chemical fixation the chromocentres of the nuclei appeared to 

 contain much more phosphatase than in freeze-dried specimens. 

 Thus in the case of kidney tissue the broad outline of the re- 

 sults obtained with chemical fixatives is correct, but the detailed 

 distribution of phosphatase cannot be studied without freeze- 

 drying. In all the tissues which I have studied all the cytologi- 

 cally satisfactory fixatives give a picture substantially similar 

 to that obtained by freeze-drying. This, however, may not be 

 the case with tissues containing large amounts of freely diffus-. 

 ible phosphatase, such as small intestine. 



The general conclusion appears to be that freeze-drying 

 should be employed wherever possible. Where this is impos- 

 sible, a wide variety of fixing agents will probably give an 

 overall picture which is correct in outline but misleading in 

 detail. 



The General Status of Techniques for the Localization 

 of Alkaline Phosphatase 

 The general conclusions which may be drawn from critical 

 studies of alkaline phosphatase techniques differ slightly with 

 the various techniques. In all cases it is desirable, as indicated 

 above, to use methods for the detection of diffusion artefacts 

 based upon the technique of imposed sections, or one of the other 

 techniques given above. When this is done, where the initial 

 precipitate is calcium phosphate, fairly accurate localization 

 of enzyme can be determined even within a nucleus, though it 



