90 HEWSON SWIFT 



problem that may be approached. There is as yet no adequate method 

 known to the author for chromosomal PNA. A quantitative color reaction 

 would increase our knowledge, as yet fragmentary, of the variations of PNA 

 in interphase nuclei and mitotic chromosomes (see Chapter 20). Although 

 DNA has been considered heterogenous by several workers on the basis of 

 enzyme or solubility studies, no cytochemical tecnhique has been described 

 that would reflect this complexity. Fractionation techniques to distinguish 

 different types of DNA on a cytochemical level would obviously be of great 

 interest. Cytochemistry is a new field, however, and in the space of com- 

 paratively few years has already provided the techniques of analysis for 

 hundreds of studies on the biology of nucleic acids. A tremendous number 

 of additional problems await investigation with the methods already at 

 hand. 



rX. Methods 



The following are intended merely as suggestions for those unfamilar with cyto- 

 chemical techniques for nucleic acids. Modifications may be necessary with some 

 tissues. 



1. Fixation 



Neutral formalin: Stock 40% formaldehyde solution 1 part; distilled water, 9 

 parts with added CaCOs • Small tissue blocks fixed for 1 to 12 hours, followed by 

 washing in water 5 to 12 hours. May be made up in saline for delicate tissues (e.g., 

 chick fibroblasts in tissue culture). 



Acetic alcohol: Absolute alcohol, 3 parts; glacial acetic acid, 1 part (must be freshly 

 made) . Small tissue blocks 1 to 12 hours, followed by 2 or 3 changes of 95% or absolute 

 alcohol, 1 hour each. Prolonged fixation may decrease basophilia." 



Carnoy's fluid: Absolute alcohol, 6 parts; chloroform, 3 parts; glacial acetic acid, 

 1 part. Treat tissues as for acetic alcohol. 



Serra's fluid: Absolute alcohol, 6 parts; stock 40% formaldehj'de solution, 3 parts; 

 glacial acetic acid, 1 part. Treat tissues as for acetic alcohol. 



2. Extraction 



Trichloroacetic acid treatment for nucleic acid removal: 5% trichloroacetic acid 

 at 90° for 5 to 15 minutes. Most acetic alcohol-fixed tissues are readily extracted. 

 Extraction is more difficult after formalin fixation, and often ineffective after fixation 

 with chromic acid fixatives. Treatment should be as short as permissible to avoid 

 protein removal. Protamines are quite readily e.xtracted. 



Ribonuclease: crystalline preparations made according to Kunitz'^^ or obtained 

 commercially may be used in concentrations from 0.1 to 2 mg./ml. Proteolytic ac- 

 tivity may be eliminated by dissolving 5 mg. of the enzyme in 1 ml. of 0.2 saturated 

 ammonium sulfate and placing in a boiling water bath for 5 minutes before making 

 up to desired concentration. The pH may be adjusted to 6 or 6.5 with 0.01 A'^ NaOH. 

 Sections may be treated for 1 to 2 hours at 37° (or room temperature). The method 

 works well with acetic alcohol-fixed and formalin-fixed tissues, but high enzyme 

 concentrations are necessary for PNA removal from chromic acid-containing fixa- 

 tives. 



