CYTOCHEMICAL TECHNIQUES FOR NUCLEIC ACIDS 91 



Deoxyribonuclease: Crystalline preparations made according to McCarty/' or 

 obtained commercially may be used in concentrations from 0.1 to 1 mg./ml., and 

 adjusted to pH 6.5 with 0.01 A^ NaOH. Magnesium sulfate (0.003 M) may be added, 

 but is usually not necessary. Treatment at 37° (or room temperature), for 30 to 60 

 minutes for most acetic alcohol-fixed plant tissues, and 1 to 2 hours for acetic alcohol- 

 fixed animal tissues, or shorter times with agitation. The enzyme fails to work with 

 many other fixatives; it can be made to work with some formalin-fixed tissues if these 

 are subjected to water at 90° before enzyme treatment. 



3. Feulgen Reaction 



Feulgen reagent: Add 0.5 g. basic fuchsin {C.I. 677) (or pararosaniline, C.I. 676) 

 to 100 ml. water at room temperature, add 1 g. potassium (or sodium) metabisulfite 

 and 10 ml. of jV hydrochloric acid. Shake at intervals until straw-colored (about 3 

 hours), add 0.25 to 0.5 g. activated charcoal, shake, filter, and store in tightly stop- 

 pered bottle in refrigerator. Reagent should be water-clear. If still yellowish add more 

 charcoal and refilter. 



Rinse: 10 ml. .V hydrochloric acid, 10 ml. 5% potassium (or sodium) metabisulfite, 

 180 ml. water. 



Procedure: Hydrolyze sections in N hydrochloric acid at 60°. Most acetic alcohol- 

 fixed tissues have a 12-minute optimum, and formalin -fixed tissues 14 minutes. Treat 

 with Feulgen reagent 1 hour, followed by 3 rinses, 10 minutes each (longer for thick 

 sections), wash in water for 5 minutes, dehydrate, and mount. If hydrolysis removes 

 sections from slides, they may be held in place with thin films of celloidon. Run slides 

 to absolute alcohol, dip briefly in 0.5% celloidon solution in absolute alcohol-ether 

 (1:1), allow to dry partially in air for about 15 to 30 seconds, then run slides to water 

 for hydrolysis. 



4. Basic Dyes 



Azure B.-"* Treat acetic alcohol -fi.xed tissues for 2 hours at 37° with 0.25 mg./ml. 

 buffered at pH 4.0 (e.g. McIIvaine buffer: 24.6 ml. of 0.1 M citric acid to 15.4 ml. of 

 0.2 M disodium phosphate). Rinse slides in water and leave in tert-hutyl alcohol for 

 12 hours; mount in balsam or plastic media. Stains DNA blue-green, PNA purple, 

 and other basophilic substances (mucin, chondroitin) reddish. 



Methyl green-pyronin: There have been numerous procedures recommended. That 

 of Kurnick^"" gives very satisfactory results with most acetic alcohol-fixed tissues 

 (see also Brachet") . Methyl green loses its specificity for DNA in some tissues after 

 formalin fixation. Methyl green {C.I. 684) or ethyl green {C.I. 685) (0.2%) may be 

 made up in water or acetate buffer (0.1 M) at pH 4.1 or 4.2. Solutions should be shaken 

 repeatedly with chloroform in a separatory funnel until the chloroform ceases to 

 show traces of color from crystal violet contamination. Kurnick recommends staining 

 for 6 minutes, although longer times are desirable for some tissues. Blot, and dif- 

 ferentiate in two changes of n-butyl alcohol (overnight differentiation in tert-hutyl 

 alcohol has also been recommended"). Counterstain in pyronin B {C.I. 741) in ace- 

 tone 30 to 90 seconds, clear in xylene, and mount in balsam or plastic media. Kurnick 

 obtained the best differentiation with n-butyl alcohol, which removes pyronin, hence 

 the necessity of separate staining solutions. Many workers have found pyronin un- 

 satisfactory for PNA. Kurnick reports that it "serves primarily as a counterstain and 

 is not found to be a reliable indicator of ribonucleic acid." For this reason Korson^" 



20" N. B. Kurnick, Stain Technol. 27, 233 (1952). 

 "' R. Korson, Stain Technol. 26, 265 (1951). 



