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Feulgen Technique

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					Feulgen Technique for DNA
Dr. Muhammad Rafique

Feulgen Technique
Stain is a staining technique discovered by Robert Feulgen in 1924 and used in histology to identify chromosomal material or DNA in cell specimens. It depends on acid hydrolysis of DNA, therefore fixating agents using strong acids should be avoided. The Feulgen reaction takes advantage of the ability of hydrochloric acid to convert DNA in fixed cell preparations to an aldehyde. This aldehyde is then identified using a Schiff reagent, which is specific for aldehydes. The combination of acid hydrolysis and subsequent aldehyde staining with a Schiff reagent constitutes the Feulgen procedure

Feulgen Technique
Schiff regent is commonly used in histology for both the Feulgen procedure and for the Periodic Acid Schiff (PAS) procedure. The difference in these two procedures is the acid used for hydrolysis, and this difference results in forming aldehydes from different cellular constituents (PAS stains glycogen). Schiff regent is commonly used in histology for both the Feulgen procedure and for the Periodic Acid Schiff (PAS) procedure. The difference in these two procedures is the acid used for hydrolysis, and this difference results in forming aldehydes from different cellular constituents (PAS stains glycogen).

Periodic acid-Schiff stain
Periodic acid-Schiff (PAS) is a staining method used in histology and pathology. This method is primarily used to identify glycogen in tissues. The reaction of periodic acid selectively oxidizes the glucose residues, creates aldehydes that react with the Schiff reagent and creates a purple-magenta color. A suitable basic stain is often used as a counterstain.

Periodic acid-Schiff stain
PAS staining is mainly used for staining structures containing a high proportion of carbohydrate macromolecules (glycogen, glycoprotein, proteoglycans), typically found in e.g. connective tissues, mucus, and basal laminae.

Feulgen Technique
Fixative:
Carnoy’S Fluid

Procedure:
Deparaffinize and hydrate to water I. 1 N- hydrochloric acid at 60° C, 6 minutes for Carnoy’s Fluid, 8 minutes for formalin or Helly’s II. Distilled water for 30 seconds III. Schiff reagent, in dark for 2 hours IV. Bleaching Solution, 3 times, 2 times each. Wipe the slide carefully each time V. Distilled water, 1 minute VI. Fast Green (FCF) 5 to 10 seconds VII. 95 % ethanol & rinse VIII.Dehydrate, clear and cover (Permount)

Result
DNA  Red Cytoplasm  Green

Solutions
I. Schiff Reagent II. 1N – Hydrochloric Acid Concentrated Hydrochloric Acid (37 to 38% assay) ------- 8 cc Distilled Water ---------------------------------------------------- 100 cc III. Bleaching Solution 1 N Hydrochloric Acid ------------------------------------------ 5 cc 10 % Aqueous Solution Sodium Metabisulfite (Na2S2O5) -- 5 cc Distilled Water ---------------------------------------------------- 100 cc IV. Fast Green FCF ---------------------------------------------------- 0.05 gm V. 95% Ethanol -------------------------------------------------------- 100 cc