296 Feulgen and PAS Technics (chap. 19) 



solutions: 



Fluorescent Schiff's reagent: 



acriflavine dihydrochloride 1 .0 gm. 



potassium metabisulfite 2.0 gm. 



distilled water 200.0 ml. 



N hydrochloric acid 20.0 ml. 



Dissolve acriflavine and potassium metabisulfite in distilled water; 

 add hydrochloric acid. Keep overnight before using. 



Periodic acid: 



periodic acid 1.0 gm. 



M/15 sodium acetate 10.0 ml. 



absolute ethyl alcohol 90.0 ml. 



Acid alcohol: 



70% ethyl alcohol 99.0 ml. 



hydrochloric acid, concentrated 1.0 ml. 



procedure: 



1. Deparaffinize and hydrate sections to water. Smears can be carried 

 directly into next step. 



2. Transfer to pre-heated N hydrochloric acid, 60°C: sections, 10 

 minutes; smears, 3-4 mintues (or 1% periodic acid: 10 mintues 

 for PAS). 



3. Wash briefly in distilled water. 



4. Transfer to fluorescent Schiff reagent: 20 miniues- 



5. Wash in acid alcohol: 5 minutes. 



6. Wash in fresh acid alcohol: 10 minutes. 



7. Dehydrate in absolute alcohol: 1 minute. 



8. Clear in xylene: 1 minute, and mount. 



results: 



DNA — bright golden fltiorescence 

 other elements — dark green to black 



comments: 



The advantage of this method over the conventional one is that 

 smaller amounts of dye molecules are more easily observed. 



Culling and Vasser warn that previotisly heated hydrochloric acid 

 is important; cold hydrochloric acid can produce negative results. 



Also timing is important; the reaction may weaken if slides are left 

 in hydrolysis too long. 



