Feulgen Nucleal Reaction
for DNA

Solutions

Hydrochloric acid, 1N
Schiff's reagent
Light green, 1% aqueous

Tissue sample
5µ 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).

Method

  1. Bring sections to water via xylene and ethanol.
  2. Rinse briefly with cold 1N hydrochloric acid.
  3. Place into prewarmed hydrochloric acid for the appropriate time at 60°C.
  4. Rinse briefly with cold 1N hydrochloric acid.
  5. Rinse briefly with distilled water.
  6. Place into Schiff's reagent for 30-60 minutes at room temperature.
  7. Give three sulphite rinses of about 1 minute each.
  8. Wash well with water.
  9. Optionally, counterstain with light green for 1 minute.
  10. Dehydrate with ethanol clear with xylene and mount with a resinous medium.

Expected results

Notes

  1. 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   Fixative Time   Fixative Time
    Bouin
    Carnoy
    Champy
    Do not use
    8
    25
    Flemming
    Formalin (NBF)
    Formal sublimate
    16
    10
    8
    Helly
    Zenker
    SuSa
    8
    5
    18

  2. 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   Fixative Time   Fixative Time
    Alcoholic fixatives
    20 minutes to 2 hours Formalin containing fixatives 35 minutes to 4 hours Formalin vapour 2-8 hours

  3. 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:–
     
    Potassium metabisulphite, 10% aqu. 5 mL
    Hydrochloric acid, 1N 95 mL

 

Reference
Pearse, A. G. E., (1968, 1972)
Histochemistry: Theoretical and Applied, Ed. 3
Churchill Livingstone, Edinburgh, London, UK

 


 

 

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