108 CYTOCHEMISTRY OF PROTEINS 



treatment with a solution of heavy metal will liberate the NH 2 

 group which may then be treated with a second cytochemical 

 reagent. A more detailed discussion of these reagents has been 

 given elsewhere (Danielli, 1949). 



Methods of Increasing Sensitivity 



Where tissue components are present in small amounts, the 

 methods given above may not be sufficiently sensitive. So far we 

 have considered three methods for increasing sensitivity. 



1. Probably the method which is potentially able to give the 

 greatest increase in sensitivity is to modify procedures of the 

 type given in preceding sections so as to obtain a fluorescent com- 

 pound. The specimens may then be studied in the fluorescent 

 microscope. A considerable gain in sensitivity should be achieved 

 even by visual inspection. If photographic means are used there 

 should be, in theory, a very great increase in sensitivity indeed. 

 On the other hand, the intrinsic fluorescence of the specimen may 

 pose some difficulties. These, however, may perhaps be met by 

 using selective quenching agents and by observing the fluores- 

 cence only in selected regions of the spectrum. Imperfections in 

 the optical equipment will, of course, also set a limit to the sen- 

 sitivity as determined by photographic methods. We have not 

 as yet paid much attention to the use of fluorescent compounds, 

 but it does hold much promise. 



2. A technique which we have found of considerable value 

 involves only a slight modification in many of the procedures 

 set out above. In these procedures colour has been obtained by 

 forming an azo dye in the section. If, in the final stage of the 

 formation of this dye, an amine is used which can be diazotised 

 after coupling, it is in theory possible to repeat the procedure, 

 linking on yet another amine, after diazotisation. Theoretically, 

 this process may be repeated an indefinite number of times. In 

 practice, however, although there is a very considerable increase 

 in the intensity of coloration produced by repeating this step a 

 small number of times, the increase is not directly proportional 

 to the number of couplings, the increment diminishing as the 

 number of couplings is increased. Consequently, we have found, 

 with an amine such as a-naphthylamine, that eight couplings are 

 the maximum which it is profitable to undertake. 



