How to Use Hematoxylin-Eosin Staining

How to Use Hematoxylin-Eosin Staining


Histology, the study of tissues at the microscopic level, plays a crucial role in understanding the structure and function of various biological specimens. One of the fundamental techniques in histology is the Hematoxylin and Eosin (H&E) staining method. H&E staining imparts color to different structures within tissues, allowing for detailed examination under a microscope. In this guide, we will walk you through the step-by-step process of using Hematoxylin and Eosin staining for histological analysis.

Hematoxylin-Eosin staining of duodenum and liver tissues. Figure 1. Hematoxylin-Eosin staining of duodenum and liver tissues. (Lin LY, et al.; 2017)

Materials and Equipment

Before starting the staining procedure, it is essential to gather all the necessary materials and equipment. Here's a list of items you'll need:

Formalin-fixed tissue sections

Hematoxylin stain

Eosin stain

Alcohol solutions (70%, 95%, and absolute)


Distilled water

Glass slides

Cover slips


Safety Precautions

As with any laboratory procedure, it's crucial to prioritize safety. Wear appropriate personal protective equipment, including gloves and a lab coat. Work in a well-ventilated area, and be cautious while handling chemicals.


  • Deparaffinization and Rehydration

Start by placing the formalin-fixed tissue sections on glass slides. Tissues are often embedded in paraffin wax for preservation, and the first step is to remove this wax. This is achieved through deparaffinization and rehydration.

a. Place the slides in xylene for 5 minutes to dissolve the paraffin wax.

b. Transfer the slides to a series of alcohol solutions with decreasing concentrations (absolute alcohol, 95%, and 70%) for rehydration. Each step should take approximately 3-5 minutes.

  • Hematoxylin Staining

Hematoxylin is a natural dye that selectively stains nuclei blue, providing contrast to other cellular structures. Follow these steps for Hematoxylin staining:

a. Immerse the slides in a Hematoxylin stain for about 5-10 minutes. The staining time may vary based on the concentration and type of Hematoxylin used.

b. Rinse the slides gently in tap water to remove excess stain.

c. Differentiate the slides in an acidic alcohol solution (1% acid alcohol) until the desired blue color intensity is achieved. This step helps distinguish between different cellular components.

d. Rinse the slides again in tap water to stop the differentiation process.

  • Eosin Staining

Eosin is a counterstain that imparts a pink color to cytoplasmic structures. This step adds contrast to the blue-stained nuclei. Follow these steps for Eosin staining:

a. Immerse the slides in an Eosin stain for 1-5 minutes. The staining time may vary depending on the concentration and type of Eosin used.

b. Rinse the slides in tap water to remove excess Eosin stain.

c. Dehydrate the slides in a series of alcohol solutions with increasing concentrations (70%, 95%, and absolute) for approximately 3-5 minutes each.

  • Clearing and Mounting

The final steps involve clearing the stained tissue sections and mounting them for microscopic examination.

a. Place the slides in xylene for 5 minutes to clear the tissues.

b. Mount the cleared slides with a mounting medium and cover slips.

  • Microscopic Examination

Allow the mounted slides to dry completely before examining them under a microscope. Use a light microscope with appropriate magnification to observe the stained tissue sections. Hematoxylin-stained nuclei should appear blue, while Eosin-stained cytoplasmic structures should appear pink.

  • Interpretation and Analysis

Now that you have successfully stained and prepared your slides, it's time for interpretation and analysis. Look for specific cellular structures, such as nuclei, cytoplasm, and connective tissue. H&E staining is particularly useful for identifying pathological changes, distinguishing between cell types, and evaluating tissue morphology.


Hematoxylin and Eosin staining is a foundational technique in histology, providing invaluable insights into the microscopic world of tissues. By following this comprehensive guide, you can successfully utilize H&E staining to enhance the visualization and understanding of cellular structures. Whether you're a researcher, student, or healthcare professional, mastering this staining technique opens doors to a deeper comprehension of biological specimens and their underlying histological characteristics.

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  1. Lin LY, et al.; Aminoazo dye-protein-adduct enhances inhibitory effect on digestibility and damages to Gastro-Duodenal-Hepatic axis. PLoS One. 2017, 12(4):e0170555.

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