DIAZONIUM HYDROXIDES 101 



view of the fact that a considerable part of the protein in a fixed 

 tissue section is in the denatured state. I shall not dwell on this 

 point further at the present time. But it should perhaps be men- 

 tioned that this problem does not really become important in 

 most cases until quantitative studies are being made. At present 

 there is so much doubt as to how far microspectrophotometric 

 observations are quantitative that it does not seem worth while 

 to go into the problems arising from masking and unmasking of 

 chemical groupings. 



Diazonitjm Hydroxides as Chromogenic Reagents 



Diazonium hydroxides will react with histidine, tryptophane, 

 and possibly nucleic acid to give azo dyes. There are, of course, 

 many other possible components of tissues with which diazonium 

 hydroxides will react, including most phenols. The most con- 

 venient diazonium hydroxides have so far proved to be tetrazot- 

 ised benzidine and dianisidine. Frequently it is found that, when 

 one of these compounds is allowed to react with a tissue section, 

 a sufficient intensity of colour is developed for cytochemical pur- 

 poses. Under these conditions the reagent is linked onto the pro- 

 tein or nucleic acid by only one of its two active groups. If, how- 

 ever, the colour is not sufficiently intense, it may be intensified 

 by washing the section to free it from excess of reagent, and 

 then placing the section in a solution of phenol or amine which 

 then couples onto the free end of the reagent. For example,* 



Pr< >OH 



Pr< >OH 



N=N< >— < >N=N-OH 



1 



Pr< >OH 



N=N< >-< >-N=N< >NH< 



* Here and elsewhere, Pr denotes protein. 



