100 CYTOCHEMISTRY OF PROTEINS 



producing absorption in the ultraviolet and infrared regions. 

 But with all regions of the spectrum, the same general problems 

 must be taken into consideration. The general requirements of a 

 chromogenic reagent are: 



1. The reagent should not give a generalized staining, i.e., it shall be 

 inert to most components of a tissue section and also it should be colour- 

 less itself. 



2. The reagent should be readily washed out of sections by inert 

 solvents. 



3. The reagent should have a known specificity, i.e., it must be known 

 just what groups it will attack. 



4. The product of the reaction between the agent and the protein or 

 nucleic acid shall be indiffusible. 



5. The reagent shall produce its effect under mild conditions. For ex- 

 ample, it is essential that none of the steps involved shall dissolve com- 

 ponents of the tissue section. 



6. It is desirable that there should be several independent methods for 

 colouring the same group, so that the results obtained by these different 

 methods may be compared. 



Many of the points concerned have been discussed in more 

 detail elsewhere (Danielli, 1946-1949) . 



Practically all the chemical reagents which are available will 

 in fact attack more than one group of a protein or nucleic acid. 

 Consequently, it is frequently necessary to use two reagents, one 

 of which will be a non-chromogenic reagent used to block one of 

 the groups which would otherwise be coloured by the other reagent. 

 In fact, it can safely be said that the development of non-chro- 

 mogenic blocking agents is a key point in the cytochemistry of 

 the proteins. 



The complication which has not as yet received consideration 

 is the accessibility of some of the chemical groups of proteins and 

 nucleic acids to the reagent being used. It is well known from 

 the work of Anson and Mirsky and others that tyrosine and SH 

 groups of proteins may be masked in a native protein, and only 

 become accessible to chemical reagents after denaturation. Simi- 

 lar observations have been made on histidine, and no doubt all 

 groups of proteins are to some extent inaccessible to chemical 

 reagents. Sometimes, as is the case with histidine, a group which 

 is accessible to a reagent in a native protein may become inacces- 

 sible in a denatured one. This question of masked groups pre- 

 sents a very difficult problem for cytochemistry, particularly in 



