The document discusses H&E (hematoxylin and eosin) staining, which is the most widely used staining technique in histopathology. H&E staining differentially colors tissue components, with hematoxylin staining nuclei blue and eosin staining cytoplasm and other tissues shades of pink. The process involves deparaffinizing tissue sections, staining with hematoxylin, differentiating with acid, counterstaining with eosin, and mounting for examination under a microscope. H&E staining allows clear visualization and analysis of cells and structures to enable histopathological diagnosis.

1. H & E STAINING –
Histopathology staining
PRESENTED BY
D. JASMINE PRIYA - B.Sc., DCA., M.Sc., PGDCLT
DR. NGPARTS 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. 1. 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