Protocol, Best Practices, Steps & More

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H&E Staining Overview: A Guide to Best Practices

For routine diagnosis, the use of Hematoxylin and Eosin (H&E) is by far preferred for viewing cellular and
tissue structure detail by pathologists. The variation of stain intensity is often driven by the pathologist’s
learning experience and personal preference. Because this stain demonstrates such a broad range of
cytoplasmic, nuclear, and extracellular matrix features, nearly all teaching texts use H&E images. We
continue to use this simple and essential stain today, which has remained unchanged for well over a
century.

The staining procedure for H&E follows a basic protocol:

Dewaxing
Dehydration
Hematoxylin
Differentiation
Bluing
Eosin
Dehydration
Clearing
Cover-slipping

The format is easily reproduced and the reagents resilient enough to allow for large numbers of slides to
be stained consistently before reagents need to be changed.

In the past, stains and their components were routinely made by the laboratory. Commercially available,
premade reagents were uncommon and expensive. So, as an economical way to perform H&Es, techs
learned to make the dyes as needed. The challenge was to make sure the quality of the stain was
consistent. Different techs follow recipes using their own individual approach, so making dyes was
typically left to one person. That said, there were also more techs in the laboratory, so the time needed
to prepare the reagents did not take away from the overall workload of the team.

Many laboratories find ordering their stains to be the easiest way to ensure consistent and repeatable
quality. A large variety of both hematoxylin and eosin stain combinations provide the ability to customize
the desired results with very little hassle. As more techs retire and companies become leaner, staffing

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decreases make the use of commercially available reagents ideal because the techs are better able to
focus on embedding and cutting, which are the areas of the laboratory that offer the least amount of
automation.

For new professionals entering the field of histology, the days of making reagents are quickly becoming
a thing of the past. I find this shift may be problematic, primarily because some of the art that is
characteristically histology is lost. One of my personal challenges when working with students is helping
them to troubleshoot staining anomalies. When stains are made by the laboratory, the team learns from
hands on experience what happens when a component is missing or inappropriately added to the
mixture. These changes can make subtle alterations in the stained slides, which the histology team is
ultimately expected to fix. Ultimately, newer histology teams can struggle when troubleshooting the
subtle changes that can be easily fixed, without having the experience of making reagents and the
lessons learned in that process.

What’s in an H&E Stain?

Hematoxylin and Eosin stains are used in many areas of the histology laboratory, including frozen
sections, fine needle aspirates, and paraffin fixed embedded tissues. To better understand what makes
a well-stained slide, it is important to understand the components of the stain.

Hematoxylin is used to illustrate nuclear detail in cells. Depth of coloration is not only related to the
amount of DNA in the nuclei but also to the length of time the sample spends in hematoxylin.

Hematoxylin is a reasonably simple dye to make. The dye itself is extracted from the tree Haematoxylum
campechianum. Oxidation of the hematoxylin produces hematein, which is the actual dye used in an
H&E stain. Addition of the mordant improves the ability of the hematein to attach to the anionic
(negatively charged) components of the tissues.

Hematoxylins are typically classified by the mordant used before staining. Mordants strengthen the
positive ionic charge of the hematin. This aids the bonding of the hematin to the anionic tissue
component, which is most commonly chromatin. The type of mordant also influences the final color of
the stained components. The most common mordant used in routine histology is aluminum ammonium
sulfate (alum). This mordant causes the nuclei to be red in color, which is then changed to the more
familiar blue color when the sample is later rinsed with a weakly basic solution.

Mayer's hematoxylin is an alum hematoxylin, a commonly used stain that may be employed for both
progressive and regressive stains. It is often used as a nuclear counterstain for special stains
and immunohistochemistry. For these applications, Mayer's is used to stain the nuclei and then blued
without the use of a differentiator. Mayer's is a water-based stain.

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Harris hematoxylin is another commonly used alum hematoxylin that may be used for progressive
staining of cytology specimens but can also be used for either progressive or regressive staining in
histology. The staining tends to provide clear nuclear detail. One challenge when using Harris is that it is
best differentiated with a mild acid, as opposed to the more commonly used hydrochloric acid-based
differentiators. Harris is an alcohol-based stain. Gill's hematoxylin is an alum hematoxylin. It may be
used as a progressive or regressive stain and is available in different concentrations. Because it is made
with water and ethylene glycol, autoxidation of the stain is typically prevented over months, making it
more stable than Harris hematoxylin. However, the nature of Gills is such that extra-nuclear staining may
occur. Mucin and even adhesives used on the slide may become heavily contaminated with Gills.

The hematoxylins that use iron salts as a mordant are typically used in special stains. This is because
they can demonstrate more tissue structures than alum hematoxylins, such as myelin and elastin fibers.
One of the best known is Weigert's, which is used in the Verhoeff-Van Gieson stain, shown in the image.

Having reviewed the nuclear stains, let’s talk about the cytoplasmic component dyes. Eosin is the most
commonly used counterstain that distinguishes between the cytoplasm and nuclei of cells. It is typically
pink, with different shades of pink for different types of connective tissue fibers.

Eosin Y is the most commonly used form of eosin and may be used in both water and alcohol. The
addition of a small amount of acetic acid will also sharpen the staining of the eosin. Eosin with phloxine
added will enhance the reds seen with H&E staining. So for those who want to see richer looking reds,
phloxine may be added.

Many laboratories find ordering their stains to be the easiest way to ensure consistent and repeatable
quality. A large variety of both hematoxylin and eosin stain combinations provide the ability to customize
the desired results with very little hassle. As more techs retire and companies become leaner, staffing
decreases make the use of commercially available reagents ideal because the techs are better able to
focus on embedding and cutting, which are the areas of the laboratory that offer the least amount of
automation.

Other eosin mixtures are sometimes used, such as EA50 and EA65. These stains are primarily used for
cytology, and in addition to eosin Y, include light green, yellowish, and Bismarck brown. The addition of
these two dyes provides for the variations in color from pale blue to pink cytoplasm, best noted in the
squamous cells of a pap smear. The concentration of the mixture determines the designation of 50 or
65.

The differentiation of stains allows for the ability to selectively remove stain from tissues to the taste of
the viewer. In the case of hematoxylin, hydrochloric acid (for rapid differentiation) and acetic acid (for
slower, more controlled differentiation) are most commonly used. While hydrochloric acid (HCl) has
historically been the standard, milder acids are being used to provide gentler dye removal. Part of this
trend is due to the use of automated staining, which must accommodate the movement of the robotic
arm in addition to the time spent in the reagent.

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Bluing reagents, such as Scott's Tap Water, are used to change the hematoxylin from red to the
traditional blue color we expect. These slightly basic solutions chemically alter the dye to produce this
color change. In some locations, the tap water contains enough minerals so that the pH causes the
water to be basic enough to allow for the bluing of nuclei without the need for a bluing specific reagent.
In most cases, though, labs typically add this step to ensure appropriate bluing.

In combination, these components make up the standard stain most used in the histology laboratory.

H&E Stain Protocol Selection

The next step to having a good stain is determining what type of H&E stain is desired. There are
typically three types of H&E stains: progressive, modified progressive, and regressive.

Progressive staining occurs when the hematoxylin is added to the tissue without being followed by a
differentiator to remove excess dye. Because there is no differentiation step, background staining can
occur, especially with charged or treated slides. Pathologists sometimes prefer this type of stain,
because the noncellular material, such as mucin, becomes stained with the hematoxylin. This
extracellular staining can be an indicator of well differentiated tumors.

The following table contains a protocol with a simple regressive stain that provides a nice balance of
nuclear and cytoplasmic stains. This protocol is designed with a mild acid differentiator in mind.

Once the staining components have been selected, it is good to start with the baseline protocol. From
there, edit either the hematoxylin in 30 second increments OR the eosin in 15 second increments.
Remember, eosin will tend to penetrate much faster. Unless there is the need to significantly lighten or
darken the eosin staining intensity, the shorter increments are best. It is also important that only one
stain is changed at a time. It may appear that the hematoxylin is overstained, when the eosin just needs
to be richer.

As laboratories continue to grow, the need for consistent results and continuous throughput is essential.
Reproducibility is an important part of laboratory stain quality. When hand staining, human variables can
make each stained slide rack look different from the last. The addition of automation not only removes
the potential for inconsistency, but also frees technologists up to perform other tasks in the laboratory.

It is important that the proper balance of the dyes is achieved. Overstaining with hematoxylin can give
the illusion of understained eosin, just as overstaining with eosin can cause the hematoxylin to appear
lighter than it actually is. So, when optimizing the stain, make sure to only edit the time of one of the
components. This technique will help eliminate the need to spend additional time adjusting the stain.

Xylene 2 minutes

Xylene 2 minutes

100% ethanol 2 minutes

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 100% ethanol 2 minutes

95% ethanol 2 minutes

Water wash 2 minutes

Hematoxylin 3 minutes

Water wash 1 minute

Differentiator (mild acid) 1 minute

Water wash 1 minute

Bluing 1 minute

Water wash 1 minute

95% ethanol 1 minute

Eosin 45 seconds

95% ethanol 1 minute

100% ethanol 1 minute

100% ethanol 1 minute

Xylene 2 minutes

Xylene 2 minutes

Coverslip

With regressive and modified progressive staining, a differentiator is used. If the differentiator is made in-
house, there is the potential for it to be either too weak or too strong. Both scenarios will impact staining.
If the differentiator is stronger than intended, it will remove more hematoxylin and will make the nuclei
pale. Time is also important. Too much time in a properly prepared differentiator will also remove more
hematoxylin and will ultimately understain the nuclei.

Mild acidity is critical to the shelf life of hematoxylin. Without it, the alkalinity of the tap water rinse will
raise the pH such that the dye lake can precipitate, and the color will change from cherry red to purple
red. Adding small amounts of acetic acid to the hematoxylin periodically will aid in maintaining
appropriate pH and can extend the life of the stain.

Water is used as a differentiator for eosin. It is common to follow the eosin step with 95% ethanol. The
ethanol aids with rinsing the slide, while water pulls excess eosin from the tissue. This step can help with
coloration control but extending the time provides for lighter stains, while shortening the time maintains
brighter coloration. However, excess water in xylene can continue the differentiation process and can be
seen after coverslipping as a pink haze on the slide.

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Not all tissues are created equally. Cysts and fatty samples, even when processed correctly, may be
very difficult to see grossly once the slide has been stained. These samples often have open spaces
where fluids or fat were in the cell, and the thinness of the cell walls may give the appearance of being
light when the coloration is simply an artifact of the tissue type.

Highly cellular samples (e.g., tonsil, lymph node) can be very concerning. Remember that lymphocytes
have little cytoplasm, and there is not nearly the cellular material between cells as with other tissues. For
this reason, the hematoxylin does not have to compete with the eosin. The compact nature of the cells
also concentrates the DNA, giving these highly cellular tissues the appearance of being overstained,
when in reality, they may simply need to be sectioned thinner.

The use of clean and fresh dewaxing reagents is essential for the removal of paraffin from the slide prior
to the addition of the dyes. While xylene is the most commonly used solvent, xylene substitutes are
gaining in popularity because they are considered less hazardous and more ecofriendly. Water in
solvents, whether from reagent contamination or a high humidity environment, reduces the ability of the
solvent to remove the paraffin. Remaining paraffin prevents the dyes from penetrating the tissues, thus
giving an uneven appearance.

The simplest way to prevent this from occurring is to change reagents more frequently. Adding a small
amount of desiccant pellets (about a tablespoon per reagent vessel) will also reduce water
contamination within solvents. These measures are especially important when using a xylene substitute,
as these reagents tend to be far less tolerant of any water contamination than xylene.

Step by Step Guide to Performing an H&E Stain

The hematoxylin and eosin stain (H&E) is the most widely used stain in histology and histopathology
laboratories. When it is properly performed it has the ability to demonstrate a wide range of normal and
abnormal cell and tissue components and yet it is a relatively simple stain to carry out on paraffin or
frozen sections. In histopathology, a high proportion of cases can be diagnosed by an experienced
pathologist using an H&E stain alone.

Small numbers of slides can be effectively stained manually, while in laboratories that have a high
throughput, staining can be performed successfully and consistently by using automated slide stainers.

There are a number of different hematoxylin and eosin formulations in popular use, each with various
advantages and disadvantages. Some laboratories prefer to prepare their own solutions whilst others
choose ready-to-use commercial products.

Introduction

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The H&E stain provides a comprehensive picture of the microanatomy of organs and tissues.
Hematoxylin precisely stains nuclear components, including heterochromatin and nucleoli, while eosin
stains cytoplasmic components including collagen and elastic fibers, muscle fibers and red blood cells.
In a high-quality H&E stain, there are subtle differences in the shades of color produced by the stains,
particularly eosin, and this aids in the detection and interpretation of morphological changes associated
with disease.

It is important that people performing and assessing H&E stains for quality are aware of the subtleties of
the stain, know what can be achieved when the stain is properly performed with high-quality reagents,
and know what to look for microscopically. The maintenance of consistent, high-quality H&E stains is a
fundamental requirement in histopathology laboratories.

In the following sections, the basic steps in performing an H&E stain are outlined.

Remove the Wax

Following the preparation of a paraffin section, all the elements are infiltrated with and surrounded by
paraffin wax which is hydrophobic and impervious to aqueous reagents. The majority of cell and tissue
components have no natural color and are not visible. The first step in performing an H&E stain is to
dissolve all the wax away with xylene (a hydrocarbon solvent).

Hydrate the Section

After thorough de-waxing, the slide is passed through several changes of alcohol to remove the xylene,
then thoroughly rinsed in water. The section is now hydrated so that aqueous reagents will readily
penetrate the cells and tissue elements.

Apply the Hematoxylin Nuclear Stain

The slide is now stained with a nuclear stain such as Harris hematoxylin, which consists of a dye
(oxidized hematoxylin or hematein) and a mordant or binding agent (an aluminum salt) in the solution.
Initially this stains the nuclei and some other elements a reddish-purple color.

Complete the Nuclear Stain by “Blueing”

After rinsing in tap water, the section is “blued” by treatment with a weakly alkaline solution. This step
converts the hematoxylin to a dark blue color. The section can now be rinsed and checked to see if the
nuclei are properly stained, showing adequate contrast and to assess the level of background stain.

Remove Excess Background Stain (Differentiate)

On most occasions when Harris hematoxylin is employed, a differentiation (destaining) step is required
to remove non-specific background staining and to improve contrast. A weak acid alcohol is used. After
this treatment, blueing and thorough rinsing is again required. Staining methods that include a
destaining or differentiation step are referred to as “regressive” stains.

Apply the Eosin Counterstain

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The section is now stained with an aqueous or alcoholic solution of eosin (depending on personal
preference). This colors many nonnuclear elements in different shades of pink.

Rinse, Dehydrate, Clear and Mount (Apply Cover Glass)

Following the eosin stain, the slide is passed through several changes of alcohol to remove all traces of
water, then rinsed in several baths of xylene which “clears” the tissue and renders it completely
transparent. A thin layer of polystyrene mountant is applied, followed by a glass coverslip. If the stain
and all the subsequent steps have been properly performed, the slide will reveal all the important
microscopic components and be stable for many years.

Troubleshooting H&E Stains

Though the H&E stain is a relatively simple stain to perform, there are a variety of artifacts that can
interfere with a good stain.

Artifacts can be attributed to a variety of causes.

Troubleshooting H&E Stains

Steps to Better Routine (H&E) Staining + Coverslipping

From patient to pathologist, preparing tissue specimens for histological examination requires care, skill
and sound procedures. This guide provides practical advice on best practice techniques and simple
ways to avoid common errors.

Tips for better routine staining and coverslipping are highlighted in this section. We hope each step
provides a valuable reminder of good histology practice and helps with troubleshooting when
unacceptable results do occur.

Use Accurate Timing

Each step in the staining protocol is accurately timed.

Step times in staining are approximate and “if we are in a hurry” some steps are skipped. This can
produce inconsistent results.

These sections were cut from the same block at the same thickness and manually stained H&E by
different staff members using what was supposed to be the same method. Even macroscopically, the
inconsistency of the stain can be seen.

Regularly Monitor Quality

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Control slides are regularly stained to monitor stain quality.

Control slides are never used for H&E stains. This can make it very difficult to determine whether a
staining problem is due to poor reagents, an inappropriate protocol or poor fixation.

This section of kidney includes a variety of eosinophilic tissue elements and well-preserved nuclei that
allows a reliable assessment of the quality of H&E staining. Placenta is another specimen type that can
be used as a useful control.

Standardize Staining Conditions

Agitation, wash and drain times are optimized for all steps during staining.

Agitation, wash and

Agitation, wash and drain times are inconsistent. Solvents and reagents rapidly become contaminated.
Staining becomes inconsistent.

One of the benefits of using an automated staining instrument is that agitation, wash and drain times are
consistent. Providing other variables are properly controlled, this will ensure good, consistent results.

Ensure Complete Dewaxing

Slide dewaxing is optimized.

Slide dewaxing is sometimes incomplete, and slides contain patches of residual wax. This produces
unstained, or unevenly stained areas in sections.

This H&E stained section shows a large unstained area on the left and several smaller areas that are
either partially stained or unstained. This is due to incomplete wax removal prior to staining.

Renew Reagents Regularly

Solvents and staining reagents are regularly replaced based on the number of slides stained or racks
processed.

Replacement of solvents and staining reagents is haphazard. They are not replaced until stain quality
declines.

This section shows poor quality, muddy hematoxylin staining. This reagent should be replaced
immediately.

Hydrate Sections Thoroughly

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Slides are thoroughly hydrated prior to hematoxylin staining.

Hematoxylin solution rapidly becomes contaminated with alcohol and sometimes xylene. This causes
uneven staining.

The uneven hematoxylin staining visible in the epidermis in this skin section was caused by residual
xylene (and traces of wax) present when the hematoxylin was applied.

Monitor Hematoxylin Quality

The performance of hematoxylin solutions is carefully monitored. During their working life, hematoxylin
solutions are progressively diluted by carry over from slides and racks and are also affected by
continuing oxidation.

Hematoxylin staining is variable from day-to-day, and no attempt is made to understand why. For
example, the staining bath surface area, the extent of aeration during staining, and the ambient
temperature can all affect the oxidation rate.

Two slides from the same control block are shown. They were stained H&E using identical protocols on
an automated stainer but with an interval of seven days between runs. Even macroscopically, the
variation in the level of staining is obvious.

Ensure Complete Nuclear “Blueing”

Thorough “blueing” of nuclei with Scott’s alkaline tap water substitute or ammonia water is always
performed after hematoxylin staining. This requirement is influenced by the natural pH of the local tap
water.

Sometimes nuclei appear pinkish in completed sections due to incomplete “blueing” in alkaline tap water
after hematoxylin staining. Nuclei that are understained with hematoxylin (or over-differentiated) and
overstained with eosin also appear pink.

A. In this section the epidermal nuclei are poorly defined and are pinkish in color. This section was not
properly “blued” in alkaline water after hematoxylin staining (skin, H&E)

B. This shows another section that was properly “blued” after the nuclear stain. Here the nuclei are
much better defined (skin, H&E).

Avoid Uneven Eosin Staining

“Blueing” is followed by a very thorough wash in tap water to remove residual alkali that can impede
eosin staining and cause weak and uneven staining.

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Inefficient washing after “blueing” (leaving residual alkali) causes eosin staining to be weak and uneven.

This section demonstrates the effect of residual alkali on eosin staining. Note the patchy nature of the
stain (spleen, H&E).

Monitor Eosin pH

The pH of the eosin solution is monitored. It is kept close to pH 5.0 to maintain optimal staining. The
addition of a couple of drops of acetic acid can be used as a convenient means of lowering pH.

No attempt is made to monitor the pH of eosin. When staining intensity falls away the solution is
replaced (carry over of alkaline tap water can cause the pH of eosin solutions to rise).

A section of lung stained H&E. The eosin stain is uniformly very weak and quite unacceptable. Note that
the only components stained with eosin are the red blood cells.

Thoroughly Dehydrate Before Clearing and Coverslipping

Sections are thoroughly dehydrated before being placed in xylene for clearing.

Sections are sometimes rushed through alcohol to xylene. Clearing in xylene contaminated with water
can result in the presence of tiny water droplets in the tissue that are seen microscopically as opaque
areas lacking detail.

This section lacks clarity (it appears opaque to the naked eye). Careful examination reveals tiny water
droplets to be present throughout

Avoid Drying and Crystal Formation

The coverslip is always applied before the section has a chance to dry and a high-quality mountant is
used. The long-term storage qualities of the mountant must be known because crystals can appear in
poor quality mountant,sometimes after a long period (months or years).

Sections are allowed to partially dry before the coverslip is applied causing some nuclei to appear black.
Mountant chosen based on price alone may develop crystals during long-term storage and coverslips
may lift.

A. This section was allowed to partially dry before coverslipping. This has caused tiny air bubbles to be
trapped over some nuclei making them appear black (sometimes referred to as “corn-flaking”). B. A
coverslipped section stained H&E showing a multitude of refractile spherocrystals that developed from
poor quality mountant within six months of mounting.

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