1. The H & E Stain
The “Routine” Stain

2. Outline of Topics in this
Presentation:
 Purpose/need to stain tissue under the microscope
 Nature of Hematoxylin dye/oxidation
 Mordant-dye-lake concept
 Different Hematoxylin formulas/mordant
 Summary of Hematoxylin staining theory
 Progressive v. Regressive staining
 Differentiation
 Bluing
 Counterstaining- the nature of Eosin dye
 Overview of H&E procedure
 Each step in more detail
 Automatic v. Hand-staining
 Quality control

3. The Need to Stain:
Visualizing Tissue Elements
 The constituents of tissue are mostly transparent
and are visible under the microscope without
staining only when extreme differences in
refractive index exist (RI of unstained tissue =
1.53-1.54)
“Biological dyes” - highlight and differentiate
tissue components and allow them to be
seen under the microscope
 H & E staining is the general tissue stain most
commonly used in all histology laboratories

4. Hematein and Hematoxylin
 Hematoxylin is a natural dye extracted from the
wood of the haematoxylin campechianum, a type
of logwood tree.
 It is sold commercially as a mixture of
Hematoxylin with other added chemicals
 The active dye is not Hematoxylin itself, but
rather Hematein
 Hematoxylin can be considered as the dye
precursor, and Hematein the oxidation product
which acts as the active dye source
 Hematoxylin can be considered a basic dye that
will stain acidic tissue components such as
nucleic acids (Basophilic)

5. Continuous Oxidation In Progress
 Hematoxylin solutions have a definite “shelf-life” and inferior
staining will result from overusing the solution. Hematein,
which further oxidizes to a dark precipitate in this continuous
process eventually exhausts the stain
 Air exposure will cause oxidation, chemical oxidizers may also
be used to speed oxidation to Hematein; such as sodium
iodate, or potassium permanganate
 You may wonder why a solution of Hematoxylin is used instead
of the active Hematein; this is so that the supply of active dye
may be replenished by the Hematoxylin and the “shelf-life” of
the solution is extended
 However despite this, there will always be some of the final
oxidation product of Hematein, which forms a dark precipitate
 This must be removed by filtering before use or it will deposit
on the final slides.

6. Mordant Required
 Hematein is a weak basic dye that requires a
mordant to help link it to the desired tissue
elements
 A mordant is a metal with a valence of at least
two. (2+ charge)
 A mordant links the dye to the tissue by means
of a covalent and coordinate bond (chelation)
 The chelate is the complex formed from a
mordant and a dye and is referred to in histology
as a lake
 Metal salts often used as mordants in histology
include; iron and aluminum

7. Chemical Structures:
Hematoxylin & Hematein
Hematoxylin Hematein
DNA and RNA stain by forming salt unions with the basic dye molecule;
This attachment is dependant on the chelate formed by dye-mordant
complex.
Note: Oxidation (loss of electron) is demonstrated by the loss of hydrogen and
its electron from the Hematoxylin structure

8. A diagram showing how a mordant can be used to
link the dye molecule to selected tissue elements
 A Mordant-Dye “lake” using aluminum
 The mordant allows attachment where otherwise
there would only be a weak affinity
 The colored property of the dye (chromophore)
allows visualization of the site under the
microscope.

9. Some Frequently Encountered Hematoxylin
Formulas and their most common uses:
 Aluminum mordant: includes Harris, Mayer’s, Erhlich’s, &
Delafield’s
 Iron mordant: Weigert’s, Lillie’s, & Heidenhain’s
 Iron Hematoxylins such as Weigert’s using a mordant such
as ferric chloride are stable only a short time due to the
oxidation strength of this mordant
 Iron Hematoxylins are used in techniques where aluminum
Hematoxylin would be decolorized by subsequent steps in
the procedure
 Aluminum Hematoxylins are most often used for routine
staining because decolorization in succeeding steps is not
an issue, and the stability is greater due to less propensity
for rapid oxidation making them more practical for
extended use

10. To Summarize:
Hematein alone has a weak affinity for
tissue elements
 For effective staining of nuclear material
with Hematein you must have:
 A mixture of Hematoxylin/Hematein-the
dye precursor and active dye to replenish
the active dye as oxidation occurs
 A mordant, such as an aluminum or iron
salt to link the dye to the nuclear material
 A solvent, such as water in which to
dissolve the dry powder dye which acts as
a carrier of the dye

11. Additional Ingredients Added to Many
Hematoxylin Solutions and their Functions
 Oxidizing agents to convert the precursor
hematoxylin to hematein
 Acids to adjust pH, to extend shelf life
 Stabilizers to control the rate of oxidation
 Additions to the solvent to retard
evaporation and precipitation
 Additions such as glycerin, which retard
over-oxidation and discourage fungal
growth

12. Progressive vs. Regressive
Hematoxylin Methods:
 Nuclear staining using Hematoxylin may be accomplished
using progressive or regressive staining techniques
 Some commonly used Hematoxylin formulas are most
often used in either a progressive or regressive fashion
 However, most Hematoxylins may be used progressively
or regressively in various techniques
Exception:
 Iron mordant Hematoxylins are often preferred for special
staining techniques; being less resistant to decolorization

13. Progressive Staining
 Progressive staining just means that the tissue is
left in the staining solution just long enough to
reach the desired endpoint
 Frequent monitoring of stain quality may be
needed to determine when staining is complete
 The staining intensity is controlled by the time it
is immersed in the solution
 An example of progressive staining is H& E
staining of frozen sections using Gill’s
Hematoxylin

14. Regressive Staining
 Regressive staining involves deliberate
over-staining where the dye completely
saturates all tissue elements
 The tissue is then selectively de-stained
using a process called differentiation
 Harris Hematoxylin is popularly used
regressively in many histology labs for
routine H & E staining
 Regressive staining is often preferred
when very clear differentiation of tissue
elements is desired

15. Differentiation is Selective De-staining:
 Differentiation is achieved by using a dilute acid, most
typically acid alcohol
 In a well-stained slide, only the nuclei, calcium deposits
and some mucins will be stained by the hematoxylin
 Differentiation is affected because the ions in the
differentiating solutions diffuse more rapidly than the dye
molecule releasing loosely attached dye
 The solution will also remove the dye more easily from
some tissue elements than others (specificity)
 Differentiation is halted by washing in water when the
desired endpoint is reached
 Note: with all Hematoxylin staining methods, progressive
or regressive, the endpoint is the same

16. Bluing
 The nuclei will be stained the purplish
color of the acid dye
 Shifting the color range to blue provides a
much better contrast to the usual pinkish-
red counter-stains
 Bluing is the process of shifting the color
from reddish to purple blue by the
application of a weak alkaline solution
 Bluing is utilized in both progressive and
regressive techniques

17. Two Bluing Methods
 1. The slides may be dipped into a weakly alkaline
solution such as ammonia water
 2. The slides may also be washed in tap water,
which may be slightly acid (pH 6.0-6.8) but this is
still more alkaline than the Hematoxylin (pH 2.6-2.9)
so bluing still occurs
 Note: differentiation of the nuclei was accomplished
by the acid alcohol and halted by previous water
rising, so bluing is not needed for differentiation of
the nuclei, only color shift

18. Next we will look at the
Eosin Counter-Stain
 Eosin counter-staining is used to
demonstrate the general architecture of
the tissue and to provide contrast to the
now stained nuclei
 Eosin is an acid dye that binds to the
basic parts of the cell, i.e. the cytoplasm
(Acidophilic)
 An optimal Eosin stain will stain the
cytoplasm and other tissue elements in
three shades of pink

19. Considerations in the Application of the Eosin
Counter-stain:
 A mordant is not required
 The endpoint is not as sharp
 The shade of the color is important to provide
optimal contrast, and it can be adjusted by
adding Acetic Acid which will make it redder
 A too highly concentrated Eosin solution (dye
density); will cause a blurring of the distinction
between nuclear and non-nuclear elements
 The solvent most commonly used solvent is 95%
alcohol, so Eosin can be decolorized slowly by
leaving the slides too long in alcohol solutions,
weakening the color (undesirable)

20. Eosin: The Chemical Structure
 The carboxylic salt present lends Eosin it’s acid character

21. Let’s review:
 We have been focusing on the dyes used
in H&E staining
 We have looked at some of the different
steps in the H&E procedure and their
functions
 Now we will review the whole staining
procedure as a whole
 We will see how all of the steps work
together to create a well-stained slide

22. Major Steps in the Routine H & E
1. De-paraffinization-(removal of paraffin wax using
xylene)
2. Hydration-(graded alcohols to water)
3. Nuclear staining-using Hematoxylin
4. Differentiation-Acid alcohol
5. Bluing-Ammonia Water
6. Counterstaining-using Eosin
7. Dehydration-(application of graded alcohol to
100% alcohol)
8. Clearing-Xylene (transition from alcohol to non-
aqueous reagents)
9. Note: Water-rising steps are not shown

23. Step 1: De-Paraffinizing
 The slides have usually been heated before
staining to help bind the tissue to the glass slide
and melt away excess paraffin
 However, some paraffin still remains and must
be removed so that the aqueous solution may
reach and penetrate tissue components
 This is accomplished by submersion in an organic
solvent such as xylene or a xylene substitute,
which dissolves and removes the remaining wax
 The xylene must then be removed by
concentrated alcohol since it is also not miscible
with aqueous solutions

24. Step 2:Hydration
 The slides are then immersed in
graduating solutions of alcohol and water
(from more concentrated to more dilute),
until they reach a point where they can
enter purely aqueous solutions
 Most staining techniques move from
100% “absolute” alcohol, to 95%, and
then can enter water since alcohol and
water are miscible

25. Step 3:The Nuclear Staining
 The slides are able to be stained with the
water-based Hematoxylin solutions, as we
have removed both the paraffin and the
xylene with the alcohol treatment
 The slides are immersed in the
Hematoxylin solution
 The staining time is established at each
laboratory

26. Step 4: Differentiation
 Following a water rinse to remove excess
dye, the slides are immersed in the acidic
differentiating solution
 The timing of immersion must be
established for each laboratory and
procedure so that optimal differentiation
is achieved
 The differentiation process is halted by a
water rinse

27. Step 5: Bluing
 Bluing is the process of shifting the color
from reddish to purple-blue by the
application of a weak alkaline solution
 Ammonia water (ammonium hydroxide +
water) is often used; alternatively tap
water may also be used if pH is suitable

28. Step 6 : Counter-stain
 Eosin is used to contrast the
cytoplasm with the nuclei
 Eosin will stain in three shades of
pink, provide contrast to the nuclear
stain, and show many cytoplasmic
and tissue elements

29. Dehydration: Step 7
 After the eosin step, an alcohol rinse
follows. This must be brief, as the Eosin
stain can be diluted by the alcohol
 95% alcohol is used first, followed by
100% alcohol
 Complete dehydration is necessary, to
remove any remaining water
 Again timing is important here, so you
don’t want to leave the slides in the
alcohol

30. Step 8: Clearing
 When all the water is removed, the slide can be
immersed into xylene
 Incomplete dehydration will result in a cloudy
appearance to the final slide
 Just as in processing, xylene is used as a
transition between aqueous and non-aqueous
 Xylene is miscible with non-aqueous mounting
media, but will cause a “cloud” to form on the
final if any water is present
 Xylene is also ideal as a transition due to it’s
refractive index, which produces a clear slide
that allows light to pass well under the
microscope
 Xylene is also serves to activate the adhesive on
cover-slippers using cover-slip tape.

31. Application of the Coverslip:
Final step
 When satisfied with the final result, the histologist finishes
the slide by applying either a glass or tape cover-slip
 Mounting media is applied over to tissue and which serves
to “glue” the cover-slip to the slide, protecting the tissue
from damage and exposure to air
 Most routine staining uses xylene as a clearant
 Alternatively there are aqueous mounting media which
allow cover-slipping directly from water.
 Aqueous mounting/cover-slipping is often used when
exposure to alcohols or xylene will remove or dilute the
stain applied to the tissue
 In all cases the cover-slip must be applied to completely
cover the tissue, removing all air bubbles and debris which
may interfere with viewing under the microscope
 The final result: a permanent microscopic “record” of the
tissue sample

32. Automatic H & E or “Hand-staining” ?
 Routine H & E staining can be accomplished by
using either an automated H & E stainer or by
manually executing the staining sequence
 Both methods can produce high-quality slides
 The method chosen by each laboratory is often
determined by laboratory staffing, time
constraints, specimen volumes and other factors
 The stain procedure and optimal timing for each
step must be established at each laboratory and
will be outlined the lab’s routine H&E staining
procedure

33. Quality Control
 The histologist is charged with monitoring the stain quality,
troubleshooting staining problems, and making adjustments
when necessary
 Many labs run an H&E control slide after daily changing of the
staining set-up to avert any problems before a large number
of slides are stained
 An understanding of the H&E staining procedure is essential
to help the histologist detect and correct problems
 With experience, the histologist will learn to judge the
desirable endpoints of both Hematoxylin and Eosin staining
under the microscope
 Use of a control slide and a quality control procedure will
assist the histologist in producing high-quality and consistent
H&E staining

34. The “Routine” Stain
 If everything has gone well, what we
should now have is a stained slide
that clearly differentiates the
nucleus and cytoplasm.
 The “routine” H &E stain can
provide very useful diagnostic
information, and is sometimes the
only slide needed for a diagnosis to
be made.

35. H & E : liver tissue

36. Resources:
 Bettelheim & March, Introduction to General, Organic & Biochemistry.
2nd
Edition, Saunders Publishing. 1988
 Sheehan & Hrapchak, Theory and Practice of Histotechnology. The C.V.
Mosby Co., 1973
 Milikin, Paul M.D.,
http://stainsfile.info/Stains/StainsFile/hematoxylin/hxintro.htm 2008
Information Prepared/compiled 3/12/2008 Joelle Weaver B.A., HTL(ASCP