Download HISTOCHEMISTRY : Feulgen Technique for DNA

HISTOCHEMISTRY : Feulgen Technique for DNA
In this histochemical technique, the section is treated such that the substance under
incestigation produces a compound for which a specific test exists. Basic fuchsin is a
magenta coloured dye which is rendered colourless when treated with hydrochloric acid
and sodium bisulphate. This bleached dye may react with aldehydes to give a new dye
with a stronger colour magenta. Such aldehydes may be produced when cellular DNA is
slowly hydrolysed on treatment with HC1 at 60 degrees Celsius on mild hydrolysis, if the
section is placed in the bleached dye, the aldehydes liberated from the DNA will react
with the dye and exhibit the magenta colour. Since both DNA and RNA are basophilic
(take up same type of stains) the Feulgen reaction allows a distinction to be made as to
which basophil material is DNA.
Materials and reagents required:
8u (micro) paraffin sections of rabbit testis. The tissue was fixed using a saturated
aqueous solution of mercuric chloride with 5% glacial acetic acid. The mercurial deposits
were then removed from the tissue by treating with iodine (to convert into mercuric
iodide which is soluble in alcohol) and washed in alcohol.
N HC1 39.3ml of conc. HC1 + distilled water up to 400ml
Schiffs reagent 1g basic fuchsin + 1.9g Sodium metabisulphite (anh.) + 15ml of N HC1 +
distilled water. Shake periodically for first 2 hours. Add 0.5g of fresh activated charcoal,
shake for 2 minutes. Filter. Store in dark and refrigerate.
H2SO3 36ml 10% Potassium metabisulphite + 30ml N HC1 up to 600ml with distilled
water. Light Green 0.25% light green in 70% alcohol.
Method
Bring paraffin section to water.
Wash in running water for 2 minutes.
Hydrolyse DNA by placing slides in N HC1 at 60oC for 8 minutes
Rinse in distilled water for 1 minute.
Place in Schiff’s reagent for 1.5 hours.
Drain and rinse in 3 lots of sulphurous acid solution for 5 minutes each.
Wash in running water for 3 minutes.
Counterstain cytoplasm if desired in light green for 1 minute. Then wash in distilled
water.
Dehydrate quickly through 70%, 90% and absolute alcohols.
Clear in xylene, and mount in DPX.
Observer the slide under the microscope and write notes on the technique followed as
well as the results obtained. Try a control run (i.e. section unhydrolysed).
Draw low and high power diagrams of the seminiferous tubules.