cytochemical techniques for nucleic acids 79 



1. Acid Extraction 



The technique of nucleic acid extraction with trichloroacetic acid, sug- 

 gested by Schneider,^-* has often been performed on tissue sections. Com- 

 plete extraction of nucleic acid has been reported after 5 or 10% TCA at 

 90° for 15 minutes. ^-^•^^" Tests for tyrosine and arginine were unaffected. ^*° 

 Nucleic acids are usually readily removed from tissues fixed in acetic alco- 

 hol or neutral formalin. With fixatives containing chromic acid, some baso- 

 philia may remain even after treatment for 1 hour. Some tissues are ex- 

 tracted more readily than others, and the effectiveness of the treatment 

 should always be tested with basic dyes or ultraviolet absorption. If the 

 Feulgen reaction is used to check for DNA removal, it should be remem- 

 bered that the HCl hydrolysis may further extract DNA remaining after 

 the trichloroacetic acid treatment. In some cases nuclei are seen to be 

 Feulgen-positive where the HCl hydrolysis is omitted, meaning that extrac- 

 tion may be incomplete, and that some trichloroacetic-hydrolyzed DNA 

 remains. 



Trichloroacetic acid extraction should be used with caution in connec- 

 tion with ultraviolet absorption measurements. Its use may markedly 

 increase the refractive index of proteins in tissue sections and thus increase 

 the nonspecific light loss. It may also alter the shape of the protein absorp- 

 tion curve in the region of nucleic acid absorption, due to oxidation of 

 phenolic groups. 



The use of perchloric acid for extraction of PNA from tissue homogenates 

 was described by Ogur and Rosen. '^^ It has been used on tissue sections by 

 several investigators. In Zenker-fixed cat and rat tissues, 10% perchloric 

 acid at 5° for 18 hours removed PNA selectively, provided frozen sections 

 were used; paraffin-embedded material was less satisfactory. ^'^ With acetic 

 alcohol-fixed rat pancreas, 6 minutes in 10 % perchloric acid at 25° removed 

 PNA from the cytoplasm, and also a 3-minute treatment removed about 

 40 % of the nucleic acid from the nucleus as determined by ultraviolet ab- 

 sorption. ^^^ Presumably this also represented PNA, since the Feulgen reac- 

 tion was undiminished until 9 minutes of treatment, after which time 

 presumably the DNA was extracted. Apparently in this material PNA 

 may be selectively removed, leaving the DNA in place, but time limits are 



•" W. C. Schneider, J. Biol. Chem. 161, 293 (1945). 



'23 A. W. Pollister and H. Ris, Cold Spring Harbor Symposia Quant. Biol. 10, 147 (1947). 



130 J. Brachet and J. R. Shaver, Stain Technol. 23, 177 (1948). 



'3' M. Ogur and G. Rosen, Arch. Biochem. 25, 262 (1949). 



"2 N. M. Sulkin and A. Kuntz, Proc. Soc. Exptl. Biol. Med. 73, 413 (1950). 



133 H. S. DiStefano, Science 115, 316 (1952). 



