Fluorescent Nucleal Reaction
for DNA

Solutions

Hydrochloric acid, 1N
Acid alcohol (1% hydrochloric acid in 70% ethanol)
Fluorescent Schiff reagent

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 Acriflavine Schiff's reagent for 30-60 minutes at room temperature.
  7. Place into 1% acid alcohol, 2 changes for about 5 minutes each.
  8. Wash well with water.
  9. Dehydrate with ethanol clear with xylene and mount with a resinous medium.

Expected results
Using a BG 12 exciter filter, and OG 4 (yellow) and/or OG5 (orange) barrier filter.
DNA fluoresces yellow.

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. It has been recommended that more concentrated hydrochloric acid applied at room temperature is preferable to 60°C in the standard Feulgen method.This may also be suitable with acriflavine Schiff's reagent. At step 3, place into 5N hydrochloric acid at room temperature for the appropriate time. Then continue on with step 4.
     
    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. The treatment with 1% acid alcohol serves to remove any dye which has attached to tissues ionically, since not all the dye may have been chemically altered during preparation of the Schiff's reagent.
  4. A fluorescent Schiff reagent made from Acridine orange may also be used, although the colour of the fluorescence may differ.

 

Reference

Culling, C F A, Allison, R T, Barr, W T, (1985).
Cellular pathology technique., Ed. 4, p. 189.
Butterworths, London, England.

 


 

 

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