CYTOCHEMICAL TECHNIQUES FOR NUCLEIC ACIDS 



55 



0.025% dye 



^« untreated 



TCA 



Fig. 1. The effect of pH and ionized groups on basic dye binding. The extent of 

 carboxyl group binding is evident when nucleic acids are removed by trichloroacetic 

 acid. Partial removal of amino groups with nitrous acid results in a marked increase 

 in dye binding by both phosphoryl and carboxyl groups. Acetic alcohol-fixed mouse 

 liver serial sections, cut at 6 m and stained for 1 hour at 30° in azure B solution; rinsed 

 for 1 hour in water at the same pH as staining bath, dehydrated with tert-huty\ al- 

 cohol, and mounted in clarite; pH adjusted with 0.1 N HCl or NaOH; 5% TCA treat- 

 ment, 15 minutes at 90°; HNO2 treatment, 15% NaN02 in 3 parts 50% ethanol to 1 

 part glacial acetic acid at room temperature for 1 hour. Each point represents the 

 mean of 5 or 10 measurements of areas 25 m in diameter made at a wavelength of 590 

 m^. Points are approximately equivalent to amounts of dye bound, but are too low 

 at higher extinctions due to the influence of metachromasy. 



compounds contain esters of sulfuric acid. 2* Such compounds are not re- 

 moved by ribonuclease or hot trichloroacetic acid. They also stain a differ- 

 ent color than PNA with many basic dyes (e.g., the thiazines) and, since 

 they are usually associated with polysaccharides, may also be distinguished 

 by comparison with adjacent serial sections on which the periodic acid- 

 Schiff reaction has been performed.^" The phosphoproteins of yolk may 

 also be basophilic, but staining is not diminished by ribonuclease.'^ In 

 general, basophilia due to such compounds, then, need not be confused with 

 nucleic acid staining. 



We can conclude that basic dye binding, if carried out at pH values near 

 4, is usually specific for nucleic acid. Control slides, however, should be 



29 L. Lison, Arch. biol. (Liege) 46, 599 (1935). 



30 R. D. Lillie, Anat. Record 103, 611 (1949). 



" J. Brachet, Quart. J. Microscop. Sci. 94, 1 (1953). 



