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Feulgen stain

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feulgen reaction, chemistry

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Feulgen stain

  1. 1. Feulgen stain
  2. 2. Feulgen stain Is a staining technique Discovered by Robert feulgen 1924 Used in histology to identify chromosomal material or DNA
  3. 3. Depend on acid hydrolysis therefore fixating agents using strong acids should be avoided The Feulgen reaction is a semi. quantitative technique If the only aldehydes remaining in the cell are those produced from the hydrolysis of DNA then the technique .is quantitative for DNA
  4. 4. The Feulgen reaction consists of two steps. Initially, acid hydrolysis is performed, usually for 8–12 min, resulting in the cleavage of the nitrogen bases and formation of aldehyde groups. The preparations are then placed in light yellow Schiff reagent (fuchsinesulfurous acid), which forms bonds with these groups. The red-violet product formed in this step is evidence of the presence of DNA RNA is not hydrolyzed by the HCl treatment and, thus, the reaction is . DNA-specific
  5. 5. Feulgen reaction mechanism& definition An aldehyde specific reaction based on the formation of a purple-colored compound when aldehydes react with fuchsinsulfuric acid; deoxyribonucleic acid gives this reaction after removal of its purine bases by acid hydrolysis; .used as a nuclear stain
  6. 6. The staining intensity is proportional to the DNA concentration . hydrochloric acid does not hydrolyze nucleic acids instantly therefore prolonged gives more intense reaction
  7. 7. From the morphological point of view, specific demonstration of DNA in cell structures at the light microscopic level is very little used nowadays. On the other hand, application of the Feulgen principles to electron microscopy have recently allowed specific DNA-staining procedures to be
  8. 8. FEULGEN STAINING PROTOCOL .Bring sections to water. .Rinse sections in M - HCL at room temperature 1 min. Place sections in preheated M - HCL at 60°c (this hydrolysis time. will vary and depends on the fixative. For formalin fixed tissues 8 .mins hydrolysis is required .Rinse sections in M - HCL at room temperature 1 min. .Transfer sections to Schiff's reagent 45 min - 1 hr. .Rinse sections in bisulphite solution x 3 washes - 2 mins each. .Rinse well in water. .Counter stain with 1% light green in 1% acetic acid 40 sec 8 Mount sections in DPX . Dehydrate, clear. RESULTS DNA Magenta --Cytoplasm Green--
  9. 9. It is possible to use an instrument known as a microdensitometer or microscpectrophotometer to actually measure the intensity of the pink . feulgen reaction for a given organelle
  10. 10. Schiff's reagent is prepared by pouring 200 mL of boiling distilled water over 1-g basic fuchsin. Shake thoroughly, cool to 50°C, filter, and add 30 mL 1N HCl to the filtrate. Cool to room temperature and add 1 g potassiummetabisulfite Allow the solution to stand overnight in the dark or until a light straw or faint pink color develops. If not completely decolorized, add 0.5 g charcoal powder, shake, filter through a coarse filter, and refrigerate in a tightly-stoppered bottle in the dark. Commercially prepared Schiff's reagent can be obtained from several companies
  11. 11. Schiff's reagent is a very sensitive means of detecting aldehydes, and can be used in a method to demonstrate deoxyribosenucleic acid (DNA) specifically, in contrast to unstained ribosenucleic acid (RNA) because the acid hydrolysis causes it to dissolve or because of the hydroxyl group present in ribose which has lost its oxygen in deoxyribose This method is the nucleal reaction of Feulgen. , and Rossenbeck
  12. 12. it would seem that two things happen during acid hydrolysis of DNA. The first is that the purine bases are removed and aldehyde groups on deoxyribose are formed. The second is that histones (protein associated with DNA) and apurinic acids (deoxyribose without the base) are progressively removed. Hydrolysis should be stopped and Schiff's reagent applied when the first is well on its way, but before the second has . removed too much of the aldehyde