HEMATOXYLIN

Properties of Hematoxylin

Uses of Hematoxylin

• H&E staining: Hematoxylin is broadly used in the Hematoxylin and Eosin staining (H&E staining), a standard process for visualizing tissue samples. By staining the cell nuclei blue or black, it delivers contrast to the pinkish hue of the eosin stain that colors the cytoplasm and other substances.
• Papanicolaou (Pap) Stain: Haematoxylin is the key constituent in Pap stain, which is used widely in cytology, especially in the screening of cervical cancer. It helps in highlighting cell nuclei, assisting in the detection of abnormal cells.
• Harris hematoxylin: It is an alum hematoxylin stain that tends to provide clear nuclear detail, which is used in progressive plus regressive staining. It requires differentiation with a mild acid instead of the generally used hydrochloric acid. Further, it’s an alcohol-based stain that is generally used in cytology & histology.
• Textile dyes: For centuries, hematoxylin has been used as a dye in textile and leather industries as it produces a range of colors. Further, it’s also used for dyeing silk, leather, and sometimes sutures.
• Writing and drawing ink: Hematoxylin has been used as a key element in the manufacturing of writing and drawing inks. Artists like Vincent van Gogh used hematoxylin-based inks in their drawings, particularly when a chrome mordant was used to stabilize the color.
• Mayer’s Hematoxylin: Mayer’s haematoxylin stains cell nuclei accurately, particularly in progressive staining, amplifying nuclear detail while conserving cytoplasmic components. Its color shifts from red to blue-violet with “blueing,” making it perfect for featuring nuclei in H&E stains. This contrast helps differentiate nuclear and cytoplasmic structures.
• Phosphotungstic acid hematoxylin : (PTAH) is a histological stain that combines hematoxylin with phosphotungstic acid, which highlight specific tissue structures. It is particularly useful in demonstrating contraction band necrosis, a key indicator in the analysis of myocardial infarction (heart attack) tissue samples.
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Eosin & Hematoxylin protocol  

Principle

Hematoxylin and Eosin (H&E) staining is a technique that combines Hematoxylin and Eosin dyes to differentiate between tissue types and identify abnormalities or irregularities. Hematoxylin stains acidic structures like nuclei blue, while Eosin stains basic structures like cytoplasm and extracellular matrix pink, providing contrast for histological analysis. 

Procedure

Slide Preparation

• Remove the wax (paraffin) from the slide by passing it through a series of xylene or similar solvents, then rehydrate the slide by passing it through decreasing concentrations of alcohol (e.g., 95%, 70%, 50%).
• Rinse the slides in distilled water for 1–2 minutes.

Hematoxylin Staining

• Stain slide in hematoxylin solution for 5-10 minutes.
• Rinse slide in running tap water for 1-2 min.
• To remove excess hematoxylin, the slide should be differentiated with a dilute acid alcohol solution (e.g., 0.5% acid alcohol).
• Place the slide in a solution that will help the nuclei appear blue (e.g., 1X PBS) for 30 sec – 1 min.

Eosin Staining

• Apply Eosin stain to the slide for approximately 30 seconds to achieve cytoplasmic contrast.
• Rinse briefly in tap water to remove excess Eosin.

Dehydration

• Dehydrate the slide through a series of increasing concentrations of alcohol.

Clearing

• Put the slide in xylene 3-4 times to remove residual ethanol and enhance tissue transparency.

Mounting

Apply 2–3 drops of H&E Mount to the lower edge of the cover-slip, then bring the slide up to its edge.

Invert the slide gently so the mounting medium contacts the slide, then complete the inversion to secure the coverslip.

Observation

• Nuclei and basophilic structures – Blue
• Cytoplasm and acidophilic structures – Light to dark red