GENERAL TECHNICAL CONSIDERATIONS 29 



Robison could be extended down to the cytological level. This 

 work of Gomori and of Takamatsu gave a great new impetus 

 to the study of the cytological distribution of enzymes. Their 

 work, however, suffered from the disadvantage that they had 

 not studied most of the major physico-chemical hazards and 

 problems which may arise in the cytological study of enzymes. 

 These problems may, as was indicated in Chapter 1, be formu- 

 lated as follows: 



1. How much phosphatase is destroyed by the experimental procedures? 

 And if phosphatase is destroyed, is it destroyed selectively at certain sites 

 in the tissue, or is it destroyed to the same extent at all the sites at which 

 it occurs? 



2. Does the precipitate of calcium phosphate indicate the true site of 

 the alkaline phosphatase, or does it merely indicate sites in the tissues 

 which have a high affinity for calcium phosphate? 



3. Is the enzyme in a fixed section in its physiologically normal position? 



Until answers can be given to these three questions, it is im- 

 possible to place any reliance on results obtained by this tech- 

 nique. This chapter will consist of a study of the methods by 

 which these questions may be answered, and a consideration of 

 some of the results which have been obtained in cases where 

 satisfactory answers can be provided to these questions. 



General Technical Considerations 



It may be noted that to obtain optimal results, i.e., results 

 with a minimum of fixation artefacts and a minimum of de- 

 struction of enzyme, the freeze-drying technique is by far the 

 best. Where freeze-drying is not available, one may fix in 80 

 percent alcohol, and then take the block after 2 hours into 

 absolute alcohol, in which the material must remain for at least 

 2 hours. After the treatment with absolute alcohol, the block 

 may be either taken down to water and used for cutting frozen 

 sections or cleared by passage through such agents as cedar- 

 wood oil, methyl benzoate, or benzene. It may then be infil- 

 trated and embedded in wax, at a temperature which should be 

 kept below 60° C. If the material is embedded, sections may 

 be cut by the usual procedures, flattened on distilled water, the 

 wax removed with xylol, after which the sections are taken into 

 absolute alcohol and then into 0.1 percent collodion, from which 



