Hematoxylin and Eosin (H&E) Staining – Principle, Procedure and Results

Hematoxylin and Eosin (H&E) staining is commonly performed on thin tissue sections prepared for examination under a bright-field light microscope. In histology and histopathology, these tissue sections are examined to observe the distribution of cells and the arrangement of tissues. Unstained tissue sections usually have very little contrast, making it difficult to observe many cellular details under the microscope.

Before staining, the tissue is processed and cut into thin histological sections which are placed on glass slides. In the routine paraffin-section method, the paraffin is first removed by deparaffinization. The sections are then rehydrated and stained with hematoxylin followed by eosin. After staining, the sections are dehydrated, cleared and mounted with a coverslip for microscopic examination.

Frozen tissue sections are prepared without paraffin embedding and require a different preparation before staining. The staining solutions and treatment times may vary depending on the laboratory protocol used.

What is Hematoxylin and Eosin (H&E) Staining?

Hematoxylin and eosin (H&E) staining is a differential histological staining technique used to distinguish cellular and tissue structures. It uses two dyes, hematoxylin and eosin. It is also written as haematoxylin and eosin staining.

Hematoxylin stains the cell nuclei blue to purple. Eosin stains the cytoplasm and many extracellular tissue components in different shades of pink. These differences in staining help in distinguishing the cellular and tissue structures under the microscope.

H&E staining is commonly used in routine histology and histopathology. It can be performed on both paraffin-embedded and frozen tissue sections after suitable preparation.

Purpose of Hematoxylin and Eosin (H&E) Staining

The following are the Purposes of hematoxylin and eosin (H&E) staining:

1. To increase the contrast between different cellular and tissue components.
2. To distinguish the cell nuclei from the cytoplasm and surrounding extracellular material.
3. To observe the size, shape and chromatin pattern of cell nuclei.
4. To examine the cytoplasm and extracellular tissue components, including collagen fibres.
5. To study the arrangement of cells and the normal architecture of tissues.
6. To identify changes in the tissue structure associated with inflammation, cellular injury and abnormal growth.
7. To help in the histopathological diagnosis of diseases, including cancer, by examining the stained tissue sections.

Principle of Hematoxylin and Eosin (H&E) Staining

The principle of hematoxylin and eosin (H&E) staining is based on the different affinities of cellular and tissue components for the two stains. The staining depends on the electrical charge of the dyes and tissue components, the pH of the staining solutions and the formation of a dye-mordant complex.

Principle of Hematoxylin Staining

Hematoxylin itself has very little staining ability. It is first oxidized to hematein, which combines with a metal mordant, usually aluminium salts (alum), to form a coloured dye-mordant complex known as a dye lake.

In acidic staining solutions, the hematein-aluminium complex is positively charged and is attracted to the negatively charged phosphate groups of nucleic acids. Nuclear chromatin has a high affinity for this complex and is stained by hematoxylin. Other nucleic-acid-rich structures, such as ribosomes and rough endoplasmic reticulum, may also take up the stain.

The nuclear staining is produced by the interaction of the hematein-mordant complex with the tissue rather than direct binding of unchanged hematoxylin to DNA.

The staining intensity is affected by the hematoxylin formulation, pH and staining conditions. In regressive staining, excess hematoxylin is removed by differentiation using a dilute acidic solution. The stained nuclei are then blued in water or a mildly alkaline solution, which changes the reddish-purple staining to blue. Progressive hematoxylin staining may not require acid differentiation.

Principle of Eosin Staining

Eosin is an acidic (anionic) dye carrying a negative charge. It binds mainly to the positively charged groups of proteins present in the cytoplasm and extracellular tissue components.

The tissue components that have an affinity for eosin are known as eosinophilic or acidophilic structures. Eosin produces pink to red staining of the cytoplasm, muscle fibres, collagen and other protein-rich structures.

The uptake of eosin depends on the pH of the staining solution and the protein composition of the tissue. At a suitable acidic pH, positively charged groups on tissue proteins attract the negatively charged eosin molecules. The amount of eosin retained varies with the types and concentration of proteins present in different tissues.

Reagents and Requirements for H&E Staining

Reagents

• Hematoxylin staining solution
• Eosin Y staining solution
• Xylene
• Absolute ethanol (100%)
• 95% ethanol
• 70% ethanol
• Distilled water
• Running tap water
• Acid alcohol (for regressive staining)
• Bluing solution
• Resinous mounting medium (DPX or Permount)

Requirements

• Prepared tissue sections on glass slides
• Staining jars (Coplin jars)
• Slide staining rack
• Glass coverslips
• Forceps
• Timer
• Light microscope
• Chemical fume hood
• Laboratory coat
• Protective gloves
• Safety goggles

Procedure of Hematoxylin and Eosin (H&E) Staining

The procedure of hematoxylin and eosin (H&E) staining using Harris hematoxylin (regressive method) for formalin-fixed, paraffin-embedded tissue sections is given below.

The staining times are based on a routine Harris hematoxylin protocol. They may vary depending on the staining solution, tissue type and laboratory protocol.

1. Fix the tissue in 10% neutral buffered formalin (NBF). The fixation time depends on the size and type of tissue.
2. The fixed tissue is dehydrated through graded alcohols, cleared in xylene and embedded in paraffin wax.
3. Cut the paraffin block into thin sections of about 4 µm using a microtome. Place the sections on clean glass slides and dry them to ensure proper attachment. The section thickness may vary from 2-5 µm depending on the tissue and laboratory method.
4. Immerse the slides in three fresh changes of xylene for 3 minutes each to remove the paraffin wax.
5. Transfer the slides into 100% ethanol for 2 minutes, followed by two fresh changes of 100% ethanol for 1 minute each. Place them in 95% ethanol for 1 minute and wash in water for 1 minute.
6. Stain the sections with Harris hematoxylin for 3 minutes.
7. Wash the slides in water for 3 minutes to remove the excess hematoxylin.
8. Differentiate the sections in 1% acid alcohol for 3-10 seconds. About four quick dips may be used during manual staining. Wash in water for 1 minute.
9. Place the slides in an alkaline bluing solution for 30-60 seconds. Wash them in water for 2 minutes.
10. Transfer the slides into 95% ethanol for 1 minute.
11. Counterstain the sections with alcoholic eosin solution for 1-3 minutes.
12. Remove the excess eosin and dehydrate the sections in 95% ethanol for 2 minutes, followed by two changes of 100% ethanol for 1 minute each.
13. Clear the sections in three fresh changes of xylene for 2 minutes each.
14. Add a suitable resinous mounting medium (DPX or equivalent) and carefully place a coverslip over the tissue section. Avoid the formation of air bubbles.

The sections should not be allowed to dry during the staining procedure. The slides are transferred between the different solutions after draining the excess liquid.

Progressive and Regressive H&E Staining

H&E staining can be performed using either the progressive or regressive method. The method depends on the hematoxylin formulation and laboratory staining protocol.

Progressive H&E Staining

In progressive staining, the tissue sections are stained with hematoxylin until the required nuclear staining is obtained. Mayer’s hematoxylin and certain Gill hematoxylin formulations can be used for this method.

Routine acid differentiation is usually not required. The sections are washed and blued as required by the staining protocol before eosin counterstaining.

Regressive H&E Staining

In regressive staining, the tissue sections are initially overstained with hematoxylin. The excess stain is then removed by controlled differentiation using acid alcohol.

The sections are washed after differentiation and treated with a bluing solution before eosin staining. Harris hematoxylin is commonly used for this method.

The hematoxylin staining time and duration of differentiation depend on the strength of the staining solution and the required nuclear staining intensity.

Results and Interpretation of H&E Staining

Staining Results of H&E Staining

The different cellular and tissue components show characteristic colours after hematoxylin and eosin (H&E) staining. The staining results of a properly stained tissue section are given below:

The cellular structures that take up hematoxylin are known as basophilic structures. These include nuclear chromatin and RNA-rich regions of the cytoplasm. Structures that take up eosin are called eosinophilic or acidophilic structures.

The staining intensity may vary depending on the tissue composition, fixation, staining solutions and laboratory protocol. Different tissues may also show variations in the shades of pink and purple.

Interpretation of H&E-Stained Tissue Sections

H&E stained tissue sections are examined under the light microscope to look at the arrangement of the cells and the architecture of the tissue. They are first looked at at low power to get an overview of the tissue architecture and the boundaries. At higher power you can then look at the size and shape of the nuclei, the pattern of the chromatin, the cytoplasm and the extracellular material.

In the skin sections the epidermis and the dermis (below it) are clearly visible. The collagen fibres in the dermis are pink. In the kidney sections the glomeruli, the renal tubules and the surrounding interstitium are all present.

Abnormal tissue sections may show enlarged or irregular nuclei, altered chromatin distribution and altered cell arrangement. There may be a marked increase in inflammatory cells in areas of inflammation. Necrotic tissue may show loss of nuclear detail and increased eosinophilia of the cytoplasm.

The sections may also contain staining artefacts, tissue folds, tears and changes due to poor fixation. All of these can affect the microscopic appearance and need to be differentiated from genuine morphological changes.

The interpretation of abnormal tissue architecture is dependent on the tissue type, the clinical history and the microscopic findings. Some abnormalities are not amenable to a specific diagnosis on H&E alone and further investigation (special stains, immunohistochemistry, molecular testing etc) may be required.

Precautions for H&E Staining

The following are the precautions of hematoxylin and eosin (H&E) staining:

• Use properly fixed and processed tissue sections. Inadequate fixation may affect the nuclear details and staining quality.
• Prepare thin and evenly cut tissue sections on clean glass slides. The sections should be properly dried and firmly attached to the slides before staining.
• Remove the paraffin wax completely using fresh xylene or a suitable clearing agent. Residual paraffin may prevent uniform staining of the tissue sections.
• Rehydrate the sections through graded alcohols and water before staining with hematoxylin.
• Use clean staining jars and properly maintained staining solutions. Filter the hematoxylin when required to remove stain precipitates.
• The hematoxylin staining time should be maintained according to the protocol. Prolonged staining may require longer differentiation.
• In regressive hematoxylin staining, differentiation with acid alcohol should be carefully controlled. Excessive treatment may remove the nuclear stain. Progressive hematoxylin usually does not require routine acid differentiation.
• Wash the sections properly after hematoxylin staining and acid differentiation to remove excess stain and remaining acid.
• Use a suitable alkaline solution for bluing. The sections should be washed thoroughly after bluing to remove the residual alkali.
• The pH of eosin should be maintained according to the staining formulation. Avoid carrying alkaline bluing solution into the eosin stain.
• Do not keep the sections in diluted alcohols for too long after eosin staining, as excessive treatment may remove eosin from the tissue.
• Dehydrate the stained sections completely using fresh absolute alcohol. Water contamination of alcohol and xylene should be avoided.
• The tissue sections should not be allowed to dry between staining steps or before coverslipping. Apply a suitable mounting medium and avoid trapping air bubbles under the coverslip.
• Wear suitable protective equipment when handling formalin, xylene and other hazardous reagents. Volatile solvents should be handled in a chemical fume hood. Dispose of the chemical waste according to laboratory safety procedures.
• A known control tissue section should be included during routine staining to check the staining quality.

Common H&E Staining Errors and Their Causes

The following are the common errors of hematoxylin and eosin (H&E) staining and their causes:

• Weak nuclear staining is seen when the hematoxylin staining time is too short or the staining solution is exhausted. Excessive differentiation, poor fixation and excessive decalcification can also reduce nuclear staining.
• The nuclei appear excessively dark after prolonged hematoxylin staining or insufficient differentiation. Thick tissue sections may also retain more stain.
• Reddish or brownish nuclei may be observed when bluing is incomplete due to a short bluing time, unsuitable bluing solution or acidic washing water.
• Weak eosin staining may be due to insufficient staining time, exhausted eosin or high pH of the staining solution. The stain may also be removed when the sections are kept too long in diluted alcohols.
• Excessive eosin staining occurs when the staining time is too long, the dye concentration is high or excess eosin is not removed during dehydration.
• Incomplete deparaffinization or improper rehydration may produce uneven staining. The sections may also show patchy staining when they are not completely immersed in the staining solutions.
• Thick regions of a tissue section usually retain more stain than thin regions, producing differences in staining intensity.
• Dark deposits may be present on the tissue due to precipitated hematoxylin. Contaminated or unfiltered staining solutions and dirty staining jars can also produce these deposits.
• Poor nuclear details may result from delayed fixation, inadequate tissue fixation or autolysis. Excessive heating during tissue preparation can also damage the nuclear structures.
• Folds and tears in tissue sections may occur due to improper microtome adjustment or poor spreading of sections during cutting and mounting.
• Chatter marks and cracks may be seen when the tissue blocks are too hard, the tissue is excessively dehydrated or the microtome is not cutting properly. Inadequate paraffin infiltration may also produce damaged or compressed sections.
• Poor adhesion to glass slides, insufficient drying or vigorous washing may cause the tissue sections to detach during staining.
• A cloudy appearance or white haze under the coverslip may be seen when dehydration is incomplete or the clearing agent contains water. Water-contaminated xylene may also cause eosin to spread from the tissue and produce a pink haze.
• Foreign tissue fragments and other particles may be present in the stained sections due to contamination from an unclean water bath, tissue-processing instruments, staining solutions or another tissue specimen.
• Air bubbles may be trapped under the coverslip due to improper placement, insufficient mounting medium or drying of the tissue section before mounting.

Applications of H&E Staining

The following are the applications of hematoxylin and eosin (H&E) staining:

• The normal structure and arrangement of cells and tissues can be studied under a light microscope.
• Tissue biopsies and surgically removed specimens are stained with H&E for routine histopathological examination.
• It is used to examine the morphology and arrangement of tumour cells for the identification of cancerous changes.
• To examine the presence and distribution of inflammatory cells in affected tissues.
• Cellular injury, degeneration and necrosis can be observed in H&E-stained tissue sections.
• Fibrosis and other changes in connective tissue are examined using H&E staining.
• Frozen tissue sections are stained with H&E for rapid examination during certain surgical procedures.
• It is used during autopsy examinations to study pathological changes in different organs.
• H&E-stained tissue sections are used in histology and pathology teaching to study the microscopic structure of tissues.
• The tissue changes in experimental and control groups can be compared by examining H&E-stained sections during research.
• The stained sections help in selecting tissue areas for further examination using special stains and immunohistochemistry (IHC).

Advantages of H&E Staining

The following are the advantages of hematoxylin and eosin (H&E) staining:

• H&E staining is simple to perform and requires only two main staining solutions.
• It is an inexpensive staining method compared to many special staining and immunohistochemical techniques.
• The nuclei, cytoplasm and extracellular tissue components can be distinguished in the same tissue section.
• It provides good contrast between different cellular structures and helps in the examination of tissue architecture.
• The size, shape and chromatin pattern of cell nuclei can be examined in properly fixed and stained tissue sections.
• H&E staining can be used for different types of tissues and is compatible with several commonly used fixatives.
• Both paraffin-embedded and frozen tissue sections can be stained using this method.
• Rapid H&E staining can be performed on frozen sections for examination during surgical procedures.
• The stained sections can be examined using a standard bright-field light microscope without requiring specialized equipment.
• It can be performed manually or using automated staining instruments for processing a large number of tissue sections.
• Properly stained and mounted tissue sections can be stored for several years without significant fading.
• It helps in the initial examination of pathological changes and the selection of tissue areas for further testing using special stains or immunohistochemistry (IHC).

Limitations of H&E Staining

The following are the limitations of hematoxylin and eosin (H&E) staining:

• H&E staining is a non-specific staining technique. It cannot identify specific proteins or antigens present in tissue cells.
• Different types of cells having similar morphology cannot always be distinguished by H&E staining. For example, T and B lymphocytes require specific markers for their identification.
• Some microorganisms are difficult to identify in H&E-stained sections, especially when present in small numbers. Special staining methods may be required for the detection of bacteria and fungi.
• Basement membranes, reticular fibres and elastic fibres are not clearly demonstrated by H&E staining. These structures require special stains for better examination.
• Most lipids are removed during routine paraffin tissue processing. Lipid deposits cannot be examined reliably in these sections using H&E staining.
• H&E staining does not specifically demonstrate certain tissue substances such as glycogen and mucins. Special stains are required for their identification.
• It provides limited information about the biochemical composition of cells and tissues.
• Some pathological conditions may show similar morphological changes under the microscope. A specific diagnosis cannot always be made using H&E staining alone.
• The staining quality depends on proper fixation, tissue processing and staining conditions. Improper preparation may affect the microscopic details of the tissue.
• Fine ultrastructural details of cells cannot be examined using H&E staining under a standard light microscope.
• H&E staining is performed mainly on fixed tissue sections. The movement and metabolic activities of living cells cannot be observed using this method.
• The tissue sections represent only a small portion of the specimen. Small or localized pathological changes may be missed if the affected area is not included in the sections.

References

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