168 Microscopic Histochemistry 



chemistry to form a highly insoluble precipitate with the 

 acid ion in question, to the incubating mixture. As the acid 

 is liberated, its ions will be trapped in statu nascendi by the 

 cation, to yield a precipitate at the site of formation. 



The precipitate formed is usually colorless and not easily 

 seen under the microscope. Therefore, it must be trans- 

 formed into a colored, easily observable compound. In the 

 case of the heavier metals the sections can be treated directly 

 with a suitable reagent. Soluble sulfides, for example, will 

 transform precipitates of Pb, Co, Fe, and Cu into blackish, 

 exceedingly insoluble sulfides. Fe and Cu can also be trans- 

 formed into the corresponding f errocyanides ( blue and red- 

 brown, respectively). When the primary precipitate is a Ca 

 salt, it can be demonstrated by the methods mentioned in 

 the section on calcium ( p. 33 ) . 



The alcoholic (or phenolic) moiety can be demonstrated 

 only if it is a thio-alcohol or a naphthol. Some thio-alcohols 

 form highly insoluble precipitates with heavy metals; naph- 

 thols can be visualized as azo dyes. 



The azo-coupling reaction is rapidly becoming the basis of 

 several excellent histochemical techniques and therefore de- 

 serves a somewhat detailed discussion. Its application to in- 

 soluble tissue phenols has been described in the section on 

 phenolic substances (p. 120). In the case of more soluble 

 substances, such as are produced by enzymatic hydrolysis, 

 a number of other important points have to be considered. 



Under suitable conditions, diazonium salts will couple 

 with aromatic amines and hydroxy compounds (and, in addi- 

 tion, with a number of heterocyclic compounds) to form 

 brightly colored, very insoluble azo dyes. Hydroxides (phe- 

 nols and naphthols ) couple optimally at an alkaline reaction 

 (pH 8 and up), whereas amines couple at an acid reaction 

 (pH 3-5). This is only a general rule; while it is true that 

 the coupling ability of phenols and naphthols rapidly de- 

 clines as the neutral point is approached, there are a few 

 types of naphthols which couple quite readily, almost at the 



