78 Microscopic Histochemistry 



(Burian,^^ Johnson and Clapp,^^ Steudel,^* H. Fischer,^^ and 

 Hunter^^ ) all agree that this coupling takes place exclusively 

 in the presence of a high concentration of caustic alkali ( car- 

 bonates, e.g., will not do). It is also specified that purines 

 substituted in the imidazole ring ( caflFeine, theobromine, nu- 

 cleotides ) will not react at all.^^' ^^ Therefore, if azo-coupling 

 in slides is attempted according to the suggestion of Mitch- 

 ell,^"^ as modified by Danielli^^ (at pH 9), no reaction can 

 be expected to take place. Actually, in Coujard shdes, carry- 

 ing marks made with gelatin alone and with suspensions of 

 guanine, adenine, xanthine, uracil, uric acid, and RNA, no 

 differentiation of any kind can be obtained. 



Second, all diazonium compounds are quite labile, espe- 

 cially at an alkaline reaction. On standing (even at 0° C), 

 they form dark-colored decomposition products which will 

 stain almost anything (e.g., a strip of filter paper or even a 

 collodion membrane ) and cannot be washed out completely. 

 When pieces of such stained material, after thorough wash- 

 ing in water and alcohol, are immersed in an alkaline solu- 

 tion of ^-naphthol, they will stain intensely purple. Tissues 

 treated by this method are stained quite diffusely, without 

 much differentiation of morphologic detail. The enterochro- 

 maffin granules are the only exception to this. They stain 

 selectively and quite intensely both after the primary and 

 after the secondary azo-coupling (the latter first recom- 

 mended by Clara and Canal in 1932).^^ There can be no 

 doubt that after acylation there is a marked decrease in the 

 staining of all tissue structures, but the contrast is not much 

 improved. It is remarkable that reactivity of the enter o- 



82. Burian, R.: Ztschr. f. physiol. Chem., 51:425, 1907. 



83. Johnson, T. B., and Clapp, S. H.: J. Biol. Chem., 5:163, 1908. 



84. Steudel, H.: Ztschr. f. physiol. Chem., 48:425, 1906. 



85. Fischer, H.: Ztschr. f. physiol. Chem., 60:69, 1909. 



86. Hunter, G.: Biochem. J., 30:745, 1936. 



87. Mitchell, J. S.: Brit. J. Exper. Path., 23:296, 1942. 



88. Clara, M., and Canal, F.: Ztschr. f. Zellforsch. u. mikr. Anat., 15:801, 

 1932. 



