CYTOCHEMICAL TECHNIQUES FOR NUCLEIC ACIDS 81 



tamination of the enzyme or buffer solution.^** Consequently, solutions of 

 enzyme should be freshly prepared before use on tissues, and proteolytic 

 activity should be destroyed by heating (for example, in a boiling water 

 bath for 5 minutes in 0.2 saturated ammonium sulfate'^"). Where prepara- 

 tions are to be used for quantitative ultraviolet studies, the effect of the 

 enzyme may also be tested on control Feulgen slides. 



The type of fixation used can greatly alter the effectiveness of ribonucle- 

 ase treatment." •'^'•'''^■"^ The enzyme usually works easily on acetic alcohol- 

 fixed tissues, at concentrations of 0.01 %. Although it will work after certain 

 chromic acid fixatives (Flemming's, Navashin's), higher concentrations oi 

 longer digestion periods must be used. After Bouin's or Benda's fixation, 

 the enzyme may not extract any appreciable PNA.'^^ Different tissues vary 

 in the ease of PNA removal. In certain cases, for example, in formalin or 

 acetic alcohol-fixed biopsies of human cirrhotic liver," ribonuclease had no 

 perceptible effect and also acted incompletely on sections of rat liver. ^^^ 

 Kaufmann et al.^*^ found that nuclear PNA is less easily digested in onion 

 root sections than PNA of the cytoplasm. This difference was attributed 

 to the fact, found in vitro, that DNA partially inhibits ribonuclease action 

 on PNA. 



Ribonuclease is effective on tissue sections over a wide pH range (about 

 5 to 8) . Buffers may seriously inhibit activity, and also particularly at alka- 

 line pH may remove PNA from control sections in the absence of en- 

 zyme. ^^"'^^^ In addition, when basic dyes are used, buffer ions may compete 

 for binding sites, as mentioned above. For these reasons enzyme solutions 

 are best adjusted with dilute sodium hydroxide to a slightly acid pH (about 

 5 5") 31,150 Since ribonuclease is heat-stable, it may be used at elevated 

 temperatures. Most rapid extraction occurs between 40 and 60°, although 

 the higher temperature in some cases may cause increased nonenzymic 

 PNA loss." 



Preparation of pancreatic deoxyribonuclease may be made according to 

 the method of Fischer^*' or of McCarty,^^ and can be further purified by 

 repeated ammonium sulfate fractionation.^*- This purification can effec- 

 tively eliminate ribonuclease contamination, but some proteolytic activity 



1" W. Jacobson and M. Webb, Exptl. Cell Research 3, 163 (1952). 



"« R. E. Stowell and A. Zorzoli, Stain Technol. 22, 51 (1947). 



1" R. Tulasne and R. Vendrely, Nature 160, 225 (1947). 



i« K. K. Tsuboi and R. E. Stowell, Biochem. et Biophys. Acta 6, 192 (1950). 



"9 B. P. Kaufmann, M. R. McDonald, H. Gay, N. Okuda, J. M. Pennoyer, and S. 



Blowney, Yearbook Carnegie Inst. 47, 144 (1948). 

 '=■« B. P. Kaufmann, M. R. McDonald, and H. Gay, Am. J. Botany 38, 268 (1951). 

 '*' F. G. Fischer, I. Bottger, and H. Lehmann-Echternacht, Z. physiol. Cheni. 276, 



271 (1941). 

 1" W. G. Overend and M. Webb, J. Chem. Soc. 1950, 2746. 



