//. CHEMICAL METHODS 



A. REQUIREMENTS 



The microscopic technique employing chemical methods depends 

 in almost every case on the direct observation of an insoluble 

 product of a microchemical reaction between the substance or group 

 whose distribution is being investigated and a suitable reagent. 

 Since the whole purpose of these methods is to visualize the presence 

 of a cellular or intercellular constituent in situ, it is essential that 

 the tissue be handled in a manner that will not permit the con- 

 stituent to diffuse or change its anatomical position during the pro- 

 cedure. The minimum requirements of the chemical method then 

 may be listed as: 



1. The preparation of microtome sections in which there has 

 been no significant alteration in the position of the constituent 

 being investigated. 



2. A reagent which is specific for this tissue constituent. 



3. A reaction between the reagent and constituent which is of 

 such a nature, and rapid enough, to obviate diffusion of the con- 

 stituent or of the reaction product. 



4. A reaction product, thus trapped in situ, which is capable of 

 being visualized. 



The frequency with which these requirements can be met is, 

 unfortunately, still very low. The problem is most difficult in the 

 case of those constituents which are diffusible in solution, e.g., 

 inorganic ions. While it is possible to prepare tissue sections without 

 the use of solutions by means of the freezing-drying technique, the 

 chemical formation of a substance in these sections for purposes of 

 visualization involves the use of a reagent in solution. One might 

 imagine that, if the interaction of the reagent in solution with the 

 ion in the tissue were rapid, the ion would be bound as an insoluble 



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