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  1. Staining Dr. Tarek Atia

  2. A. Decalcification Bone specimens as well as calcified tissues are the most type here. The calcium must be removed before embedding to allow sectioning. A variety of reagentshave been used to decalcify tissue such as mineral acids, organic acids, EDTA, and electrolysis.

  3. Strong mineral acidssuch as nitric and hydrochloric acids • Strong acids will remove large quantities of calcium at a rapid rate, butthey will cause damage of cellular morphology.

  4. Organic acids such as acetic acid and formic acid. However, they act more slowly on dense cortical bone. EDTA can remove calcium safely, but it works slowly, it penetrates tissue poorly, it is expensive in large amounts.

  5. Hematoxylin and Eosin (H&E) • The Hematoxylin is a basic dye that stains acidic components of cells a blue color. • This characteristic is known as basophilia. • Hematoxylin stains the nuclei of cells, and the RER of the cytoplasm. • Eosin is an acidic dye that stains the basic components of the cells a reddish-pink color. • This characteristic is known as acidophilia. • Most of the cytoplasm of cells is stained by eosin. • Bone matrix is also stained by eosin.

  6. Before staining, paraffin sections should be first deparaffinizated by Xylene. • If sections are Zenker-fixed, remove the mercuric chloride crystals with iodine and clear with sodium thiosulphate or hyposulphate.

  7. Periodic acid-Schiff (PAS) staining • PAS is a widely used staining technique that stains the neutral sugars of glycosaminoglycans a pink color. • Common components stained positively with PAS include glycogen, mucus, muco-protein, glycoprotein, the basal lamina, organ capsules, blood vessels, etc.

  8. Osmium Tetroxide • Osmium is used to stain lipids a dark black color. • It is very useful for demonstrating the myelin of myelinated nerves, or lipid droplets in the liver or steroid-secreting cells.

  9. Toluidine blue Toluidine blue is a so-called metachromaticstain. It is a blue stain that stains specific components of tissues a purple color. This change in staining known as metachromasia. Metachromasia is seen in the matrix of hyaline cartilage, or in the granules of mast cells.

  10. Impregnation Impregnation is a staining technique in which blocks of tissue are processed in solutions containing metals such as silverorgold, which attached to specific components in tissues.

  11. The silver or gold are then further processed (reduced) and develop into dark metallic deposits. The stained blocks are then sectioned.  Silver impregnation is widely used to stain neuronsand to demonstrate reticular fibers.

  12. Vital staining Vital staining refers to uptake of dyes by living cells. If we inject Trypan blue into experimental animals, the dye is rapidly engulfed by specific macrophages. We can use such vital staining to demonstrate the Kupffer cells of the liver.

  13. Supravital stain • Staining living cells outside the body (Reticulocytes = immature RBCs). • Brilliant cresyl blue stain, is good to diagnose Reticulocytosis (increase the number of immature RBCs).

  14. Giemsa stain There are a variety of "Romanowsky-type" stains with mixtures of methylene blue, azure, and eosin compounds. Among these are the Giemsa stain and the Wright's stain (or Wright-Giemsa stain). The latter is utilized to stain peripheral blood smears.

  15. Collagen - Trichrome Stain • Purpose: Used to differentiate between collagen fibers and smooth muscle in tumors, and the increase of collagen deposition in chronic diseases such as liver fibrosis. • Routine stain for liver and kidney biopsies. • Principle: As the name implies, three dyes are employed selectively staining muscle, collagen fibers, fibrin, and erythrocytes. • Results: Nuclei: Black Cytoplasm, muscle, erythrocytes: Red Collagen: Blue/ green

  16. Examples of trichrome stain: • Malory trichrome: stains collagen deep blue • Masson`s trichrome: stains collagen green Masson`s trichrome Malory stain

  17. Reticular Fibers - Gordon And Sweet's Method • Purpose: A silver impregnation technique that demonstrates reticular fibers. Reticulum is abundant in liver, spleen, and kidney. Reticulum forms characteristic patterns in relationship to certain tumor cells. • Principle: The tissue is oxidized, then sensitized with the iron alum, which is replaced with silver. The silver is reduced with formalin to its visible metallic state. • Results: Reticular fibers: Black Nuclei: Red

  18. Elastic Tissue Fibers - Van Gieson Technique • Purpose: This stain is useful in demonstrating atrophy of elastic tissue in cases of emphysema, and the thinning and loss of elastic fibers in arteriosclerosis, and other vascular diseases. • With increasing age, changes such as splitting and fragmentation occur, these changes are most obvious in the skin which becomes wrinkled and rather 'loose-fitting'.

  19. Elastic Tissue Fibers - Van Gieson Technique • Principle: The tissue is stained with a regressive hematoxylin, consisting of ferric chloride and iodine. • Results: Elastic fibers and nuclei: Black Collagen: Red Other tissue elements: Yellow

  20. Orcein Orcein staining is used to stain elastic fibers a dark brown-purple color. This is used, for example, to show the elastic components in the walls of arteries, or in the matrix of elastic cartilage.