H-E Stain

Understanding the H-E Stain: A Comprehensive Guide


Histology and pathology have greatly benefited from the Hematoxylin and Eosin (H&E) stain, a fundamental technique that facilitates the examination of tissues’ structural features. This blog will delve into the principle, components, sample requirements, test procedure, result interpretation, and precautions associated with the H-E stain method.

Principle of H-E Stain

H-E Stain plays a pivotal role in biology, histology, pathology, and cytology by revealing the intricate morphological details of tissues and lesions. It operates on the principle of differential affinity between cell components and specific dyes. The nucleus, with its acidic nature, readily binds to the basic dye Hematoxylin, causing it to display a distinct blue-violet color. Conversely, the cytoplasm and other elements, being more alkaline, take on varying shades of red when stained with the acid dye Eosin. As a result, the H&E stain provides a comprehensive view of tissue structures.

Main Components

The H-E stain method requires several reagents for successful execution:

  • Hematoxylin stain (Harris): Contains Hematoxylin, a basic dye.
  • Eosin stain (Aqueous): Comprises Eosin, an acid dye.
  • 1% hydrochloric acid ethanol solution: Combines hydrochloric acid and ethanol.
  • Dilute lithium carbonate solution: Contains lithium carbonate.

Sample Requirements

For optimal staining results, ensure that smears and sections are adequately fixed before proceeding with the staining process.

Test Procedure

Follow these steps to perform the H-E stain:

  • For deparaffinization: immerse paraffin sections successively in xylene I and II for five minutes each. After rinsing with absolute ethanol, rinse with 95% ethanol, then 80% ethanol, each for one minute. One minute of rinsing with tap water is recommended.
  • Hematoxylin Staining: Apply Hematoxylin stain (Harris) for 5 to 10 minutes. Rinse with tap water for 1 to 2 minutes.
  • Acid Treatment: Briefly immerse tissue in 1% hydrochloric acid ethanol solution, followed by tap water rinse.
  • Blueing: Apply Dilute lithium carbonate solution for 30 seconds, then rinse with water.
  • Eosin Staining: Immerse tissue in Eosin stain (Aqueous) for 30 to 60 seconds. Rinse with water.
  • Ethanol Dehydration: Dip tissue in 95% ethanol I and II for about 10 seconds each.
  • Absolute Ethanol Dehydration: Dehydrate tissue in absolute ethanol I and II for 1 to 2 minutes.
  • Xylene Transparency: Make tissue transparent by immersing it in xylene I and II for 1 to 2 minutes.
  • Sealing and Microscopic Examination: Seal with neutral resin and examine under a microscope.

Result Interpretation

After the H-E stain procedure, nuclei appear in shades of indigo blue, while cytoplasm, stroma, and fibers exhibit varying degrees of red coloration.


  • Hematoxylin Storage: Avoid cold storage and freezing of Hematoxylin stain (Harris) to prevent precipitation and redness.
  • Date-Dependent Coloring: Due to oxidation variations, perform staining tests on each unsealed bottle of Hematoxylin to adjust staining times.
  • Storage Temperature: Store Hematoxylin staining solution at 10°C to 30°C in the dark to prevent excessive oxidation.
  • Regular Filtering: Remove oxide film and aluminum sulfate crystals before use. Blueing is achieved using a Dilute lithium carbonate solution.
  • Temperature Sensitivity: Adjust dyeing time for Hematoxylin when temperatures are low.
  • Color Separation: Control differentiation time in 1% hydrochloric acid alcohol solution to ensure clear nuclear and cytoplasmic contrast.
  • Eosin Decolorization: Avoid over-immersion of eosin-stained samples in ethanol to prevent excessive decolorization.
  • Consistent Storage: Maintain proper reagent storage conditions, avoiding extreme temperatures and light exposure.
  • Timely Fixation: Timely and complete fixation of samples contributes to successful H-E stained sections.
  • Reagent Purity: Regularly replace 80% ethanol, 95% ethanol, and absolute ethanol to ensure their purity.
  • Professional Use: Use the reagent as instructed by professionals, following hygiene and safety guidelines.
  • Waste Disposal: Properly dispose of waste following hospital or environmental protection regulations.
  • Packaging Information: Refer to the outer packaging for the production date, batch number, and expiration date.


The Hematoxylin and Eosin stain method remains a cornerstone in the realm of biology, histology, pathology, and cytology. Its ability to provide a detailed view of tissue structures makes it an invaluable tool for pathological diagnosis, research, and teaching. Adhering to the outlined procedure and precautions ensures accurate and reproducible staining results, enabling a deeper understanding of the biological world.

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