Haematoxylin and Eosin Staining histopathology.ppsx

**Haematoxylin and Eosin (H&E) Stain in Histopathology**

The H&E stain is the most widely used staining technique in histopathology, providing a fundamental tool for visualizing tissue architecture and cellular morphology. Haematoxylin, a basic dye, stains acidic structures (basophilic) such as nuclei, ribosomes, and rough endoplasmic reticulum, imparting a blue-purple hue. Eosin, an acidic dye, stains basic structures (eosinophilic) like cytoplasmic proteins, collagen, and red blood cells, resulting in a pink-red color. Together, they create a high-contrast image, enabling pathologists to differentiate between cell types, identify abnormalities, and diagnose diseases.

H&E staining is essential for routine tissue examination, offering a clear view of tissue structure, cellular details, and pathological changes. It is used in diagnosing cancers, inflammatory conditions, and infections, and serves as a baseline for further specialized staining. The simplicity, reliability, and cost-effectiveness of H&E make it indispensable in histopathology laboratories worldwide. Its universal application underscores its importance in both research and clinical diagnostics.

1. HAEMATOXYLIN AND
EOSIN STAINING
M JALAL
BS MLT
HISTO-TECHNIQUE
KMU HIS KOHAT

2. OBJECTIVE
Introduction to Haematoxylin and Eosin Staining
Preparation Haematoxyin Stain
principle
Preparation of Eosin
procedure
Result

3. INTRODUCTION TO HAEMATOXYLIN AND
EOSIN STAINING
•Definition
•Hand E stain is the principal stain used for the routine tissue section staining.

4. PRINCIPLE
•Principle
•Staining technique involves electrostatic attraction b/w ions of opposite charge.
•Haematoxylin is a complex with aluminium slat is cationic and act as a basic dye.
•It is positively charged and react with negatively charged nucleic acids in the nucles.
•Eosin is anionic and acts as an acidic dye. It is negatively charged and react with positively
charged acidophilicomponents such as amino group of proteins in cytoplasm.

5. PREPARATION HAEMATOXYLIN STAIN
•Preparation Haematoxylin Stain
•Haematoxylin = 6g
•Absolute alcohol = 300ml
•D/w = 300ml
•Glycerol = 300ml [prevents rapid evaporation]
•Glacial acetic acid = 30ml [more precis and selective nuclear stain
•Potassium alum = in exess [act as a moderant]

6. PREPARATION HAEMATOXYLIN STAIN
•Preparation Haematoxylin Stain
•Haematoxylin is the crude extract from tropical log wood.
•1 Dissolve haematoxylin in absolute alcohol and add the act in the order mentioned in
side.
•2 keep the stain in a loosely pluged bottle in a keep where daylight is available.
•3 oxidation of heamatoxylin take place b/w one to two months. It is called ripening.

7. PREPARATION OF EOSIN
•Preparation of Eosin ; Eosin aqveous solution
•Stock solution ; Eosin Y - 5mg
• D/W alkaline top H2O = 100mg
•Working solution ; stock solution = 1 part
• D/W water = 4 part
•If a deeper tone is desired add 5drops of glacial acetic acid to 100ml of solution.

8. PROCEDURE
•Procedure = deparaffinization, hydration ,staining , differentiation, blueing, dehydration,
clearing,
•Absolute alcohol -- 1 mint
•90% alcohol --- 1 mint
•70% alcohol -- 1 mint
•Wash in top H2O -- 1 mint

9. PROCEDURE
•Haematoxylin-- 10 – 20 mint
•Wash in tap water -- 1 mint
•Differentiation in 1% alcohol 5 to 30 second
•Rinse quickly in tap water
•Blueing in tap water / 1% lithium
•Carbonate solution 5ml

10. PROCEDURE
•Eosin 2 mint
•Absolut alcohol -1 --- 2 second
•Absolut alcohol -2 -- 2 second /
•Xylene 1 --- 2 second
•Xylene 2 --- 2 second
•Mount with DPX/ coverslip

11. RESULT
•Nuclei,cytoplasmic rna, calcium salt --- blue
•Muscle, reratin, fiberin – bright red
•Collagen, reticulin, amyloid – pink
•Red blood cells -- orange