Histopathology staining method

Histopathology staining method

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  H & ESTAINING – Histopathology staining PRESENTED BY D. JASMINE PRIYA - B.Sc., DCA., M.Sc., PGDCLT DR. NGPARTS AND SCIENCE COLLEGE COIMBATORE
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  STAINING IN HISTOPATHOLOGY Staining is widely used in histopathology and diagnosis, as it allows for the identification of abnormalities in cell count and structure under the microscope.  A huge range of stains is used in histology, from dyes and metals to labeled antibodies.  Certain stains change the coloration of cells and tissues significantly, different from the color of the original dye complex, a phenomenon known as metachromasia.  For staining, paraffin sections of tissue are normally used.  They are rehydrated and then made translucent (cleared) using a clearing substance such as xylene, before being stained.  When observing a tissue sample under the light microscope it is often difficult to distinguish between different cells and tissue, as they are almost colorless.  Therefore staining is used to create differential coloration, allowing clearer observation and analysis of cells.
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  1. HAEMATOXYLIN ANDEOSIN (H & E): ROUTINE STAIN  Hematoxylin and Eosin (H&E) staining is the most widely used staining technique in histopathology.  As its name suggests, H&E stain makes use of a combination of two dyes, namely hematoxylin and eosin.  This combination deferentially stains various tissue elements and make them easy for observation.  This is the most common histologic stain, used to differentiate different tissue structures.  It also plays an important role in the diagnoses of various pathologies.  In the H&E stain, a mixture of oxidized hematoxylin known as hematein is used.  Due to poor affinity of hematin with tissues, a mordant is incorporated in the H&E stain.  Most commonly used mordants are salts of aluminium , iron and tungsten. This substance is known as hemalum.  When applied to a tissue section, hemalum stains nuclei blue.  The sample is then counterstained using a solution of eosin (either alcohol or water), which stains proteins and cytoplasm varying shades of pink.  Eosin are xanthene dyes and have different types, but generally Eosin Y is commonly used.
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  HAEMATOXYLIN EOSIN- Y
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  PRINCIPLE  The principlebehind H & E stain is the chemical attraction between tissue and dye.  Hematoxylin, a basic dye imparts blue-purple contrast on basophilic structures, primarily those containing nucleic acid moeties such as chromtatin, ribosomes and cytoplasmic regions rich in RNA.  An acidic eosin counterstains the basic elements such as RBCs, cytoplasm, muscle and collagen in varying intensities of pink, orange and red.
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  REQUIREMENTS 1. Harris hematoxylinstain A= 1 gm hematoxylin in 10 ml ethanol. B=20gm ammonium alum in hot distilled water. Mix a & b boil and add 0.5 gm of mercuric oxide and filter. 2. Eosin solution Yellow eosin =1 gm Distilled water = 80ml Ethanol =320 ml Glacial acetic acid = 2 drops 3. 0.5% HCL 4.Dilute ammonia water
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  Procedure  Deparaffinization: flamethe slide on burner and place in the xylene. Repeat the treatment to remove the wax.  Hydration: Drain xylene and hydrate the tissue section by passing through decreasing concentration of alcohol baths (100%, 90%, 80%, 70%) and water.  Nuclear Staining: Stain in hematoxylin for 3-5 minutes.  Wash in running tap water until sections “blue” for 5 minutes or less.  Differentiation: selective removal of excess dye from the section). Dip in 1% acid alcohol (1% HCl in 70% alcohol) for a few seconds.  Blueing: Rinse in running tap water. Dip in ammonia water until the sections become blue, followed by tap water wash.
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  • Counterstain: Stainin 1% Eosin Y for 10 minutes. • Wash in tap water for 1-5 minutes. • Dehydration: Dehydrate in increasing concentration of alcohols. • Clearing: Put slides in two xylene baths for clearing. • Mounting: Mount in DPX or other mounting media. • Observe under compound microscope.
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  RESULT AND INTERPRETATION Nuclei : blue, black  Cytoplasm : Pink/purplish pink  Muscle fibres : deep red  RBCs : orange red  Calcium : Dark blue  Mucin : Grey blue MICROSCOPIC OBSERVATION- H & E STAINING