Things to Note When Using Hematoxylin-Eosin Staining

Things to Note When Using Hematoxylin-Eosin Staining

Hematoxylin and Eosin (H&E) staining is a widely used histological technique that plays a crucial role in the field of pathology and biomedical research. This staining method provides a way to visualize and differentiate various tissue structures under a microscope, aiding in the identification of cellular morphology, tissue architecture, and pathological changes. When performing Hematoxylin and Eosin staining, there are several important factors and considerations that researchers and laboratory technicians should keep in mind to ensure accurate and reliable results.

Hematoxylin-eosin staining of skin and subcutaneous tissue showing the inflammatory infiltrate in groups NLB. Figure 1. Hematoxylin-eosin staining of skin and subcutaneous tissue showing the inflammatory infiltrate in groups NLB. (Andrade TA, et al.; 2011)

Understanding the Basics of H&E Staining

Before diving into the details, it's essential to have a fundamental understanding of how H&E staining works. Hematoxylin is a basic dye that stains cell nuclei blue-purple, while Eosin is an acidic dye that stains cytoplasm and extracellular matrix pink. This differential staining allows for the visualization of various tissue components.

Tissue Fixation:

Proper fixation of tissue samples is the first crucial step in the H&E staining process. Fixation helps preserve tissue structures and prevents degradation. Formalin fixation is a commonly used method, but the choice of fixative may vary depending on the specific requirements of the study.

Tissue Processing:

After fixation, tissues need to go through a series of processing steps, including dehydration, clearing, and infiltration with paraffin wax. Careful processing ensures the removal of water from the tissue and allows for the embedding of the specimen in a solid medium for sectioning.

Section Thickness:

The thickness of tissue sections is a critical parameter in H&E staining. Thin sections (usually around 4-6 micrometers) allow for better penetration of stains and improved microscopic visualization. Inconsistent section thickness can lead to uneven staining and unreliable results.

Proper Slide Preparation:

Slides should be clean and free of contaminants before applying the tissue sections. Adherence of sections to the slides is essential to prevent loss during subsequent staining steps. Additionally, slides must be labeled accurately to avoid confusion during analysis.

Hematoxylin Staining:

Hematoxylin is the primary nuclear stain in H&E staining. The concentration, staining time, and pH of the hematoxylin solution are critical factors. Overstaining or understaining can affect the clarity of nuclear details. It's advisable to follow standardized protocols for hematoxylin staining to ensure consistency across experiments.


After hematoxylin staining, tissue sections are typically subjected to a differentiation step. This process removes excess stain and is crucial for achieving proper contrast between the nuclei and the background. The duration of differentiation should be carefully monitored, as over-differentiation can lead to loss of nuclear detail.

Eosin Staining:

Eosin is applied after differentiation to stain cytoplasmic components and other structures. The concentration and staining time of the eosin solution must be optimized for consistent and reliable results. It's important to note that prolonged exposure to eosin can result in overstaining, affecting the overall quality of the stained sections.

Dehydration and Clearing:

Following Eosin staining, the tissue sections undergo dehydration to remove water and are then cleared to enhance transparency. The choice of dehydration and clearing agents, as well as the duration of these steps, should be carefully considered to avoid artifacts and maintain optimal tissue integrity.

Mounting Medium:

Proper mounting of stained slides is crucial for long-term preservation and microscopic examination. The choice of mounting medium can impact the clarity and stability of the stained sections. Coverslips should be applied carefully to avoid air bubbles and ensure uniform distribution of the mounting medium.

Quality Control:

Regular quality control measures, such as the use of positive and negative controls, are essential to monitor the performance of the staining process. This helps identify and rectify any issues that may arise, ensuring the reliability and reproducibility of results.

Microscopic Examination:

The final step involves examining the stained sections under a microscope. Proper calibration of the microscope, appropriate magnification, and attention to detail are crucial during this phase. A systematic approach to analyzing the stained sections helps in accurately interpreting the cellular and tissue features.


Accurate documentation of the staining process, including reagent concentrations, staining times, and any deviations from the protocol, is essential. This information is valuable for troubleshooting, replication of experiments, and maintaining a comprehensive record of laboratory procedures.

Staining Artifacts:

Researchers should be aware of common staining artifacts that may occur during the H&E staining process. Artifacts such as uneven staining, precipitation of stains, or air bubbles under coverslips can impact the interpretability of results. Understanding these potential issues allows for proactive measures to minimize their occurrence.

Adaptation for Specialized Studies:

While H&E staining is a versatile technique, researchers may need to make adjustments for specific studies or unique tissue types. For example, certain tissues may require modified staining protocols or the use of additional stains to highlight specific structures or pathological changes.

Training and Standardization:

Proper training of laboratory personnel is paramount for consistent and reliable H&E staining results. Standardizing protocols and ensuring that all team members follow established procedures contribute to the reproducibility of experiments and the generation of high-quality data.

Environmental Conditions:

Environmental factors, such as temperature and humidity, can influence the staining process. Maintaining stable conditions in the laboratory helps ensure reproducibility and consistency in H&E staining.

Waste Disposal:

Proper disposal of reagents and waste generated during the staining process is crucial for laboratory safety and environmental compliance. Follow local regulations and institutional guidelines for the disposal of chemical waste.


In conclusion, Hematoxylin and Eosin staining is a fundamental technique in histology and pathology, providing valuable insights into tissue morphology and pathology. By paying careful attention to each step of the staining process and considering the factors mentioned above, researchers can enhance the reliability and reproducibility of their results, contributing to the advancement of scientific knowledge and medical diagnostics.

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  1. Andrade TA, et al.; The inflammatory stimulus of a natural latex biomembrane improves healing in mice. Braz J Med Biol Res. 2011, 44(10):1036-47.

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