Test Catageory
فئة التحليل
Histopathology
Refrence Books
كتب مراجع
1. Bancroft’s Histopathological Techniques
Skills Required
المهارات المطلوبة
1. Histology
2. Staining
Video Resources
مصادر فيديو
Aim
For demonstration of Deoxyribonucleic acid (DNA)
Principle
The Feulgen Reaction involves a mild acid hydrolysis step, where 1N HCl at 60°C is used to break down the purine-deoxyribose bond in DNA12. This process exposes aldehyde groups12. These aldehyde groups then react with Schiff’s reagent, resulting in a magenta-colored product that indicates the presence of DNA32. This reaction is specific to DNA, as RNA is not hydrolyzed by the HCl treatment1. Therefore, the Feulgen Reaction is a valuable tool in histology for the specific and sensitive detection of DNA in cell specimens
Solutions
1-1M HCL (Hydrolysis):
Concentrated HCL…………. 8.5 ml
D.W…………………………. 91.5 ml
2-Schiff’s reagent:
Basic Fuchsin……………………….……… Stain
D.W ……………………………….……… Solvent
Potassium metabisulphite………………….. Reducing agent
Conc HCL………………….……….……… Maintain PH
Activated Charcoal………….…………..…. Remove any excess Sulpher
3-1% Light green (counter stain)
Sample Type
1. Cell culture monolayers: These are layers of cells that cover a surface.
2. Imprints (touch preparations): These are samples obtained by touching a piece of tissue to a slide.
3. Smears from fine needle aspiration biopsies (FNAB): These are samples obtained by using a thin, hollow needle to extract tissue or fluid.
4. Smears from exfoliated cells: These are cells that have been shed from the body.
5. Cytocentrifuged preparations from ascites, urine, liquor, and other body fluids: These are samples prepared by spinning down cells from body fluids.
6. Tissue sections from formalin-fixed, paraffin-embedded tissue: These are thin slices of tissue that have been preserved and embedded in paraffin for microscopic examination.
Procedure
1. Bring section to water.
2. Oxidize in 1% P.A for 5 min.
3. Wash well in different changes of D.w
4. Place in Schiff’s reagent for 15 min.
5. Wash in R.T W for (5-10) min.
6. Stain nuclei with M.H and bluing.
7. Wash in water, rinse in absolute alcohol.
8. Clear and Mount.
Results
DNA: red- purple.
Cytoplasm: green.
Notes:
1. Bouin’sfixative is not suitable as it causes over hydrolysis.
2. Decalcification, strong inorganic acid should be avoided, and organic acid For short time can be used.
3. Optimum hydrolysis time should be recommended.
4. The hydrolysis time is important, and the correct time for the fixative must Be used.
5. The 1M HCL should be preheated to 60˚C.
6. 5 M HCL at room temperature may be used but it need longer time that at 60 °c.
Soha Alnoor
suha@mlsgaate.com
A specific staining technique for DNA identification in cell specimens using Schiff’s reagent after mild acid hydrolysis.
