Histopathology staining method

H & E STAINING METHOD

1. H & E STAINING – Histopathology staining PRESENTED BY D. JASMINE PRIYA - B.Sc., DCA., M.Sc., PGDCLT DR. NGP ARTS AND SCIENCE COLLEGE COIMBATORE

2. 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.

3. HAEMATOXYLIN AND EOSIN (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.

4. HAEMATOXYLIN EOSIN- Y

5. PRINCIPLE The principle behind 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.

6. REQUIREMENTS 1. Harris hematoxylin stain 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

7. Procedure Deparaffinization : flame the 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.

8. Counterstain : Stain in 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 .

9. 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