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H922 34: Cell Biology Outcome 4: H and E Staining of Tissue Sections

H and E Staining of a Fixed Tissue Section

To stain a tissue section with Haematoxylin and Eosin to investigate the
structure of tissues

The preparation of permanent, stained sections of animal tissue is an
important technique in modern pathology. It allows normal tissue to be
distinguished from abnormal tissue. It involves the following stages:
1. Selection

6. Blocking-out

2. Fixation

7. Sectioning

3. Dehydration

8. Staining

4. Clearing

9. Mounting

5. Wax impregnation
These procedures are carried out routinely in Histology Laboratories for
research and diagnostic purposes. They are very time consuming and the
operation is, therefore, mostly automated using a tissue processor. With the
aid of an automatic tissue processor the entire process can be completed
within 24 hours; equally there are several points where the process can be
stopped and restarted as time permits.
Time constraints, and Health and Safety considerations, do not allow you to
carry out the entire process, but you will have the opportunity to see a
demonstration of some of the procedures and to stain and mount tissue
sections that have been prepared for you.

H922 34: Cell Biology Outcome 4: H and E Staining of Tissue Sections

Explanation of the stages of tissue processing

1. Selection
The piece of tissue selected must come from the correct area for the
investigation required. The size of the piece selected should be as small as
possible without jeopardising the success of the investigation itself. A small
piece of tissue has a large surface area compared to its volume and therefore
the time required to process the tissue will be kept to a minimum.
2. Fixation
Fixatives cause both physical and chemical changes within the tissue and
preserve it against subsequent treatments. A good fixative will preserve the
tissue with as life-like an appearance as possible.
A fixative must be able to penetrate into the centre of the tissue and then react
with it. Formalin is a commonly used fixative that causes cross-links to be
formed within the protein molecules in the cells.
3. Dehydration
Water must be removed from the tissue before wax impregnation. The
concentrations of alcohol used depends upon the delicacy of the tissue. With
delicate tissue dehydration might begin in 50% alcohol while for routine tissue
the starting point is normally 70% alcohol.
4. Clearing
Since alcohol does not mix with molten wax the alcohol in the tissue must be
displaced by another reagent; this must be miscible with both alcohol and
molten wax. Such reagents are known as clearing agents and the one often
used is Histoclear.
5. Wax impregnation
Displacement of clearing agent by molten wax is known as impregnation. It is
essential that this is complete since if any clearing agent remains then the
wax will remain too soft once it has cooled. For routine purposes the wax used

H922 34: Cell Biology Outcome 4: H and E Staining of Tissue Sections

has a melting point of 56C and impregnation should be carried out at a

temperature of about 58C. Ideally, once cooled, the hardness of the wax
should match that of the tissue.
6. Blocking out
This refers to pouring molten wax into a small mould and then quickly
transferring into it a piece of wax impregnated tissue. Once done, the mould is
cooled rapidly in iced water so that the block hardens quickly.
7. Sectioning
Sections are cut on a microtome using a very sharp knife. Rotary microtomes
are normally used in laboratories although they are very expensive. Rocking
microtomes can be used for occasional sectioning but the sections are of a
poorer quality.
Sections of human tissue are normally cut at 5 m in thickness. Once cut, the
sections are transferred to the surface of warm water that allows the wax to
soften (but not melt!). This allows the sections to stretch and lose the wrinkles
caused by compression of the wax block during sectioning.
Each section is mounted by floating it onto a clean slide. Once the excess
water has drained, the sections are treated in an oven at 60C for 1 hour to
attach the tissue to the slide.
8. Staining
Stains are applied to tissues to make them visible or to detect the presence of
particular chemical groups. The stains which you will be using are
Haematoxylin and Eosin (H & E). With H & E the nuclei will stain blue and the
cytoplasm pink.
9. Mounting
The purpose of a mountant is to enable the final preparation to be examined
most satisfactorily. Mounting media are either aqueous or resinous (nonaqueous) substances and the selection made depends upon the previous

H922 34: Cell Biology Outcome 4: H and E Staining of Tissue Sections

stages of preparation. You will be using a resinous medium. These are

generally a form of polystyrene in a suitable solvent, e.g. DPX or Optimount.
The tissue processor
This consists of a platform supporting a number of beakers containing
processing solutions required for the following stages:Fixation

e.g. formalin
e.g. 70%, 90% and 100% (absolute) alcohol
e.g. Histoclear or xylene

Wax impregnation e.g. Paraffin wax or paraplast

These are arranged in a circle surrounding a central arm that supports the
tissue baskets and moves them from one reagent to the next at times
determined by a clock. Small pieces of tissue can normally be processed
within 24 hours.

Automated Tissue Processor


H and E Staining
H and E staining is a routine staining procedure carried out to examine basic
tissue architecture. Haematoxylin is a basic dye that will bind to acidic
structures in the cell such as nucleic acid. In acidic conditions the dye
appears red. Sections are first stained with the haematoxylin dye and then

H922 34: Cell Biology Outcome 4: H and E Staining of Tissue Sections

exposed to a n alkaline solution to blue the dye. This procedure is known as

blueing. If done correctly nuclei should appear blue at the end of the
Haematoxylin staining can either be progressive or regressive. In progressive
staining the slide is left in contact with the dye for just the right amount of time.
In regressive staining the slide is over stained and then subsequently
differentiated using acid alcohol which washes out the excessive dye.
Eosin is the final stain used in this procedure. Eosin is an acidic dye that
binds to basic components in the cell most notably proteins. In this way the
Haematoxylin and the Eosin complement each other by binding to different
components within the cell.


Safety Notes
The solvents used in this experiment are flammable. When not in use keep
the lid on the containers.
The dyes used in this experiment are harmful and will stain. Ensure a lab coat
and gloves are worn at all times. The Omnimount is harmful so care should
be taken when using this chemical.
Carry out this procedure in a well ventilated room.
Equipment List
Per group (3-4 students):
3 x Coplin Jars containing:

100% Ethanol


70% Ethanol

H922 34: Cell Biology Outcome 4: H and E Staining of Tissue Sections

4 x Dropper Bottles containing:

Cover Slips

Harris Haemotoxylin

Stage Micrometer

Eosin Stain

Per Student

Scotts Tap Water Substitute

Paraffin Embedded Tissue Section

1% Acid Alcohol


Staining rack and bowl

Eye piece Graticule

Omnimount Mounting Media

Day 1 Staining the Tissue Section
1. Name and date your slide on the same side as the tissue section is
2. De-wax and rehydrate slides
a. Immerse the slide in Histoclear II for 5 minutes
b. Transfer the slide to 100% ethanol for 3 minutes
c. Transfer the slide to 70% ethanol for 2 minutes
d. Rinse the slide with tap water
3. Stain with Harris Haematoxylin and Eosin
a. Lay the slide with the tissue section facing upwards on the
staining rack
b. Drop Harris Haematoxylin solution onto the tissue section unitl it
is covered. Leave for 15 minutes.
c. Rinse off the dye using tap water.
d. Cover the section with Scotts Tap Water substitute to blue the
section for 2 minutes
e. Differentiate by holding the slide at an angle and dropping the
solution onto the slide so that the acid alcohol runs off
immediately. Add 5 10 drops.
f. Rinse in tap water
g. Re-blue the section by covering the slide in Scotts Tap water
substitute for 2 minutes
h. Rinse with tap water
i. Counter stain with Eosin by covering the section with a few
drops of eosin solution for 1 minute
j. Rinse in tap water

H922 34: Cell Biology Outcome 4: H and E Staining of Tissue Sections

4. Dehydrate, Clear and Mount

a. Immerse the slide in 70% ethanol for 2 minutes
b. Transfer the slide to 100% ethanol for 3 minutes
c. Transfer the slide to Histoclear II for 5 minutes (or until ready to
d. Add a drop of Omnimount to the tissue section. Slowly lower a
cover slip over the Omnimount at an angle. Try and avoid any
air bubbles
e. Leave to dry
Day 2 Examining the Slides under the microscope
1. Calibrate your eye piece graticule using the stage micrometer up to 40
x magnification
2. Examine your stained tissue section up to 40 x magnification
3. Make a labelled drawing of your stained tissue section at all
4. Using the eyepiece graticule make some measurements from your
tissue section. Identify what you have measured on your drawing.
5. Include a scale bar on your drawing.
6. Dispose of your Microscope slide correctly.

H922 34: Cell Biology Outcome 4: H and E Staining of Tissue Sections

Histoclear II (HS-202), Harris Haematoxylin (HS-400) and Omnimount (HS110) are available from National Diagnostics.
Formalin Fixed Paraffin Embedded tissue sections are available from
Microtechnical Services Ltd for 0.75/slide. A full list of the available sections
is available at their website
Eosin Stain:
1 g Eosin Y dissolved in 100 mLs of deionised water.
1% Acid Alcohol
2.7 mLs of 36% HCl + 70 mLs of ethanol made up to 100 mLs with deionised
Scotts Tap Water Substitute
20 g of MgSO47H2O + 2 g of NaHCO3 made up to 1 L with dH2O.