Feulgen Nucleal Reaction
for DNA
Materials
- Hydrochloric acid, 1N
- Schiff’s reagent
- Light green, 1% aqueous
Tissue Sample
5 µ paraffin sections of neutral buffered formalin fixed tissue are suitable. Many other fixatives are satisfactory. Fixatives containing strong acids should be avoided as this method depends on the acid hydrolysis of DNA, and acids in some fixatives may pre-hydrolyse the tissue (picric acid in Bouin’s aqueous formal-picric-acetic mixture, for example).
Protocol
- Bring sections to water via xylene and ethanol.
- Rinse briefly with cold 1N hydrochloric acid.
- Place into prewarmed hydrochloric acid for the appropriate time at 60°C.
- Rinse briefly with cold 1N hydrochloric acid.
- Rinse briefly with distilled water.
- Place into Schiff’s reagent for 30-60 minutes at room temperature.
- Give three sulphite rinses of about 1 minute each.
- Wash well with water.
- Optionally, counterstain with light green for 1 minute.
- Dehydrate with ethanol, clear with xylene, and mount with a resinous medium.
Expected Results
- DNA – red
- Background – green
Notes
- The appropriate time in hydrochloric acid varies depending on the fixative. The times given below are in minutes, but should be considered a guide only. Trials should be conducted to determine the optimum.
Fixative Time Bouin Do not use Carnoy 8 Champy 25 Flemming 16 Formalin (NBF) 10 Formal sublimate 8 Helly 8 SuSa 18 Zenker 5 - A modification of this reaction uses more concentrated hydrochloric acid at room temperature. At step 3, place into 5N hydrochloric acid at room temperature for the appropriate time. Then continue on with step 4. This variant is considered to produce darker staining and a smaller loss of DNA.
Fixative Time Alcoholic fixatives 20 minutes to 2 hours Formalin containing fixatives 35 minutes to 4 hours Formalin vapour 2 to 8 hours - Sulphite rinses are now considered unnecessary. They were used originally in the belief that placing directly into water would recolour the Schiff’s reagent and give false positive staining. It is now known that this is not so, providing the Schiff’s reagent is completely washed off. Sulphite rinses consist of:
Material Amount Potassium metabisulphite, 10% aqu. 5 mL Hydrochloric acid, 1N 95 mL
Safety Note
Prior to handling any chemical, consult the Safety Data Sheet (SDS) for proper handling and safety precautions.
References
- Pearse, A. G. E., (1968, 1972)
Histochemistry: Theoretical and Applied, Ed. 3
Churchill Livingstone, Edinburgh, London, UK
