CHAPTER V

Immunohistochemistry
 Acknowledgements
• Addisa Ababa University
• Jimma University
• Hawassa University
• Haramaya University
• University of Gondar
• American Society for Clinical Pathology
• Center for Disease Control and Prevention-Ethiopia
 Learning objectives

• Upon the completion of this chapter, the student should be

able to:
1. Explain the difference between immunohistochemistry and
immunocytochemistry.
2. Define polyclonal and monoclonal antibodies.
3. Discuss methods of antigen retrieval.
4. Explain methods of antibody staining.
5. Explain methods of antibody detection
5.1 Introduction
• Immunocytochemistry is a method used to detect a specific
antigen in the tissue to identify the type of disease.
• In these techniques, an antibody is used to link a cellular
antigen specifically to a stain that can be more readily seen
with a microscope.
• Detection of antigens in tissues is known as
Immunohistochemistry, while detection in cultured cells is
generally
• termed immunocytochemistry. For both, there is a wide range
of specimen source, antigenavailability, antigen antibody
affinity, antibody type, and detection enhancement methods.
• Thus optimal conditions for immunohistochemical or
immunocytochemical detection must be determined for each
individual situation, dependent on the above variables.
Antigen
• Antigen may be protein, carbohydrate, and lipid or a
combination.
• On the antigen molecule there are specific sites for
attachment of antibody molecules.
• The antibody attaching sites on antigens are known as
antigenic determinants or epitopes.
Antibodies
• Antibodies are classes of immunoglobulins. There are five
classes of antibodies: namely IgG,IgA, IgM, IgE, and IgD.
Immunogloblin G (IgG) is the most common and most
frequently used antibody for immunocytochemistry.
• Each immunoglobulin is composed of light chain and two
heavy polypeptide chains linked by a disulphide bond to form
a Y shaped structure.
• The terminal region of each arm varies in their amino acid
sequence and is known as variable domain.
• The variability in amino acid sequence is responsible for a
particular epitope.
• The particular epitope enables the antibody to bind
specifically to the particular antigen for which the antibody is
raised.
• The amino acid chain on the variable domain forms a cavity.
The cavity is geometrically and chemically complementary to
a single type of antigen epitope. The complementary antigen
• and antibody are held together by a combination of hydrogen
bonds, electrostatic forces andVan-deer Walls forces.
Polyclonal antibodies
• The antibodies react major with corresponding antigen and
also react minor with similar antigen.
Monoclonal antibody
• The antibody reacts specifically with its corresponding antigen
and does not react with other antibody even with antibody
having minor viabilities.
5.2 Production of Polyclonal antibodies
• An antigen of interest is introduced in to the animal.
• The animal will mount an immune response to produce
antibody.
• The B-cells will proliferate to produce numerous clones of
plasma cells.
• Each clone of cells produces an antibody with slightly different
specificity to a variety of epitope present on the antigen.
• Some of these antibodies may cross react with other
molecules and therefore they have to be removed by
absorption with appropriate antigen.
N.B: Animals may have many other antibodies present in the
serum. Theses antibodies may lead to the problem of cross-
reactivity or non-specific staining. However, if the required
• antibodies are present in high concentration, many unwanted
reactions can be eliminated by dilution.
5.3 Production of monoclonal antibody
• Monoclonal antibodies are produced by hybridoma
technique.
• When antigen is introduced in to the body; B-cells proliferate
to produce plasma cells.
• Plasma cells produce antibodies against specific antigens. By
fusion of normal plasma cells and myeloma cells (cancer cells)
a hybrid cell can be produced.
• The hybrid plasma cell is an immortal cell and produces
specific antibodies.
• The hybrid plasma cell can be grown and multiplied in cell
culture or ascitic fluid and it can produce an unlimited
number of antibodies. By careful screening, hybrids producing
antibodies of interest, i.e., of single epitope can be produced
that do not cross-react with other molecules.
• Fixation strengths and times must be optimized so that
antigens and cellular structures can be retained and epitope
masking is minimal.
• Requirements for fixation can vary widely between tissues.
For example, when using antibodies to probe for
neurotransmitter substances, most tissues must be either
immersion fixed with a mixture of glutaraldehyde and
paraformaldehyde, or with paraformaldehyde alone.
• Both acetone and methanol (precooled to -20 0C) have been
used successfully as fixatives for frozen tissue in other
instances.
• The next consideration for immunological staining is the type
of section to use. For immunohistochemistry, the common
options are fixed or unfixed cryostat (frozen) sections, fixed
“wet” or vibritome sections, or fixed, paraffin-embedded
sections.
• The choice of section is determined by a number of issues,
including the time and skill of the investigator.
• Because of the ease of use, fixed frozen sections are often
quickest and easiest to use.
• However, because of their superior fidelity, clarity, and
preservation properties, fixed paraffin-embedded tissues have
become the ultimate standard of immunohistochemistry in
histology and pathology, and anytime where archiving of
immunohistochemical information is required.
Today, there are two types of Cryostat sections:
1. Fresh, or unfixed sections where quickly frozen (snap frozen)
tissues are first cut, theneither air-dried or fixed prior to staining
and

2. Fixed frozen tissue, where the tissue is first fixed then cryo-
protected with sucrose or other stabilizer (to stabilize the tissue
cell structure) prior to freezing and sectioning.
• The advantages of frozen sections are that they allow
excellent antigen preservation, they are typically faster to
perform, and they offer flexibility, since any fixative can be
used, thereby facilitating the optimization of fixative for each
antigen.
• However, frozen sections give less morphological detail and
resolution than other methods
Sample protocols for Cryostat sections - Fresh Frozen (then
fixed) Tissue Sections:
1. Snap-freeze small tissue blocks (5x5x3 mm) in liquid
nitrogen.
2. Transfer to cryostat and cut thin (5-30 μm) sections.
3. Collect specimens on clean poly-L-lysine-coated glass slides
and dry at room temperature overnight (if you want to stain
the same day let air-dry for 1-2 hours. until completely dry).
Thorough drying is essential for good adhesion to the slides.
4. Fix sections in acetone or absolute ethanol at 4 0C for 15
minutes. Use fresh ethanol or acetone for every 10-15 slides
for best results. The organic solvents absorb moisture from the
air and tissue, as they do so, they lose their ability to fix the
tissue effectively.
5. Thoroughly air-dry at room temperature or on mild heat (30-
370C). It is during this stage that much of the chemical fixation
is being finalized. Improper air-drying will lead to “soft”
sections and likely loss of proper reactivity.
6. Proceed with immunostaining or freeze.
Fixed, frozen tissue sections
1. Fix tissue either by perfusion with fixative or by immersion in
fixative for a set time period. Most commonly, 4%
Paraformaldehyde (PFA) solutions are used.
2. Fixed tissue is then prepared for cryoprotection by
submerging the target tissue in a hydro stabilizing solution.
• The cryoprotection is complete when the target tissue no
longer floats in the stabilizing solution. Because it works well
and is relatively inexpensive, phosphate buffer saline (PBS) +
sucrose solutions ranging from 10% w/v (less protection), to
30% (w/v) sucrose (greater protection) are often used.
3. Once stabilized, tissues can be removed from the protectant
solution and frozen at -70 0C until sectioned.
4. Via cryostat (5-40 μm), where sections can be collected
directly onto slide, or floated onto slides via a PBS/water bath.
• Usually up to three sections per slide can be placed. Each
spaced well apart. The spacing prevents reagent mixing
between samples.
• Individual skill and tissue type will determine the thickness of
the sections. Sections between 10-15 μm provide the best
results for clarity and integrity. Sections between 6-9 μm tend
to tear during cutting, resulting in rough edges that can
increase the background. Thicker sections while stronger
during handling can be more difficult to stain.
5. Sections on slides are thoroughly air/heat dried on a slide
warmer, usually overnight or at least 2-3 hours at 40-50 0C.
6. Prepared slides can be stored dry at -70 0C until stained.
Equilibrate to room temperature and briefly re-dry prior to
rehydration and staining.
• The largest proportions of samples used in immunostaining
are embedded in paraffin because it provides excellent
morphological detail and resolution. Modern “paraffin” is
typically a mixture of paraffin wax and resin.
• It is an excellent embedding medium because it can be heated
to liquid state, and dissolved by xylene for infiltrating the
tissue, and then relatively quickly turned to a solid state again
for maximum structural support during sectioning.
• Typically, small blocks of tissue (10x10x3 mm) are fixed for up
to 24 hours.
• The most common fixatives used in paraffin sections are
formalin-based. These fixatives are well tolerated by the
tissues and achieve good penetration.
• The blocks are then infiltrated and embedded with paraffin
and 5-10 μm sections are cut in ribbons and mounted on
slides.
• Once mounted, the slides can be stored indefinitely until
immunostaining is required, then the paraffin must be
removed from the tissue to allow the water-based buffers and
antibodies to penetrate.
Sample Protocol for Paraffin-embedded Sections
• A. Conventional deparaffinization and dehydration sequence:
1. Incubate sections in Xylene: 2 to 3 changes, 5 minutes each.
2. 100% absolute ethanol: 2 changes, 3 minutes each.
3. 95% ethanol: 2 changes, 3 minutes each.
4. 80% ethanol: 3 minutes.
5. 50% ethanol: 3 minutes.
6. Rinse with distilled water, PBS, or Tris buffer: 2 changes, 3
minutes. each.
Note: Once sections have been rehydrated, do not allow them to
dry.
B. Place slides in pre-warmed (37 0C) 0.1% trypsin in PBS for 5-60
min. or 0.4% pepsin in 0.01N HCl for 30 minutes to one hour.
Follow by rinsing with distilled water.
C. If peroxidase conjugate is used; endogenous peroxidase
should be blocked at this stage.
• Peroxidase activity results in the decomposition of hydrogen
peroxide (H2O2). It is a common property of all hem-proteins
such as hemoglobin, myoglobin, cytochrome and catalase.
• Suppression of endogenous peroxidase activity in formalin-
fixed tissue entails the incubation of sections in 3% H2O2 for
8-10 minutes. Methanolic H2O2 treatment (1 part 3% H2O2
plus 4 parts absolute methanol) for 20 minutes. can also used,
but it is not recommended for specimens where cell surface
markers are to be stained.
• Methanolic treatment may also detach frozen sections from
their carrier glass.
D. Wash twice with PBS.
E. Proceed with immunostaining procedure (see section 5.5)
5.4 Antigen Retrieval can be achieved through:
– Enzyme digestion;
– Microwave;
– Autoclaving or pressure-cooking.
• To facilitate the immunological reaction of antibodies with
antigens in fixed tissue, it may benecessary to unmask or
“retrieve” the antigens through pretreatment of the
specimens. There are many forms of antigen retrieval
(sometimes called antigen recovery), and different antigens
and different antibodies will require different antigen retrieval
methods.
• Antigenretrieval has been shown to increase reactivity of the
majority of antigens in tissues.
• The use of antigen retrieval in immunocytochemistry is less
common. However, depending upon the particular
antibody/antigen combination it can be performed on cell
preparations, although the length of time and intensity is
typically much less than for tissue. Antigen retrieval includes a
variety of methods by which the availability of the antigen for
interaction with aspecific antibody is maximized.
• The most common techniques are enzymatic digestion or heat
induced epitope retrieval (HIER) through microwave irradiation,
autoclaving or pressure-cooking.
5.4.1 Enzymatic Digestion
• This technique involves dewaxing, rehydrating, and rinsing the
specimen in running water.
• The specimen is then equilibrated with the appropriate buffer
and incubated with aproteolytic enzyme at 37 0C, or at room
temperature. Enzymes used include pronase (0.05% (w/v) in
PBS), trypsin (0.05% (w/v) in PBS with 0.1% CaCl2) and pepsin
(0.05% (w/v) in 2NHCl).
• The conditions of concentration, time and temperature must
be controlled, so that the enzymes can break some of the
bonds formed during fixation, uncovering antigenic sites, but
the antigen should not be digested completely.
• The enzymatic activity is stopped by placing the specimen in
cold buffer (4°C) prior to processing with antibody. These
methods should be considered for some antigens/tissues.
However, proteolytic enzymes can abolish the reactivity of
some antigens.
5.4.2 Microwave irradiation
• Microwave irradiation of formalin-fixed, paraffin-embedded
specimens in buffer has been found to markedly enhance the
retrieval of antigens.
• During this procedure the energy provided helps break some
of the bonds formed during fixation, thus increasing the
number of positive cells available, and the intensity of
reactions although the exact mechanism is unclear.
• It is important to monitor the sections during the micro
waving process, to prevent damage and drying.
• Consistency of conditions between experiments including
buffer volumes, irradiation times, and microwave unit used,
will result in less variability in staining results.
• The number of samples that can be treated by microwave
irradiation at one time is limited. Typically, specimens in some
buffer are heated either at full or partial power for a few
minutes.
• Periodically, the heating is stopped and liquid is replenished.
After a set time, the solution containing the slides is allowed
to cool to room temperature slowly, then the slides are rinsed
in PBS and used for staining.
5.4.3 Autoclaving
• In order to standardize the procedure, it is important to start
with standard volumes of preheated solutions.
• After adding the specimens to the boiling retrieval solution,
the autoclave or pressure cooker should be brought to full
pressure as quickly as possible and the heating times
measured exactly from this point.
• At the end of the heating time (usually 1 to 2 minutes) the
pressure should be released. As soon as possible the hot
buffer should be flushed out with cold water. (Sections should
not be allowed to dry.) The specimens should then be washed
in buffer.
• Although the most critical feature of both microwaving and
autoclaving is probably the heating of the tissues, the pH and
composition of the solutions used are also important in the
unmasking of antigenic sites.
• Studies have found no significant difference between microwave
and autoclave treatment, but there are significant differences based
on the solutions used. Some of the buffer solutions commonly used
are 0.01 M citrate buffer (pH 6.0), 0.1 M Tris-HCl (pH 8.0) and 1 mM
EDTA (pH 8.0), with citrate buffer used most commonly.
• It should be noted that many more specimens could be treated at
any one timeusing an autoclave or pressure cooker than using a
microwave oven.
• However, preservation of the cytological detail may be slightly
inferior in sections that undergo pressure-cooking.
• A more mild procedure that can be used on many tissues is a
simple incubation in citric acid buffer, pH 3.0 (2.1 grams Citric
Acid added to 400 mL of addH20.
• Adjust to pH 3.0 with acetic acid if above 3.0, or NaOH if
below 3.0, make up to one liter final volume with distilled
H20) for 30 minutes, at 37 0C after blocking but prior to
primary antibody addition.
• Rinse slide in PBS or TBS pH 7.4 prior to staining.
5.5 Antibody Staining
• Primary antibody may be directly labeled with an enzyme (such
as horseradish peroxidase or alkaline phosphatase) or
fluorophore (such as FITC or rhodamine), or unlabeled, with
detection by a labeled secondary antibody or more complex
detection system.
• If a secondary antibody is used, it must be generated against
the immunoglobulins of the primary antibody source, e.g., if the
primary antibody is raised in rabbit, then the secondary
antibody could be goat anti-rabbit.
• The optimal titer of both the primary and secondary antibody
should be determined for each batch.
• For staining of tissue sections, it is customary to incubate with
25-50 μl of diluted antibody.
• The volume used must be sufficient to completely cover the
tissue, and to ensure that the tissue will not dry out during
incubation. Incubation times may range from 30-90 minutes
at 370C, from one to six hours at room temperature, or
overnight at 40C.
• Incubation times should be optimized empirically for each
antibody/antigen combination.
General Protocol for Immunohistochemical Staining with Polyclonal Rabbit or
Monoclonal Mouse Primary Antibody:
• The following general protocol is intended for use as a guideline in developing
antibodyspecific procedures. Different antibodies and tissues may require changes
to this procedure.
• Review of individual product datasheets and relevant literature references may be
helpful in customizing this procedure for specific applications.
1. Gently rinse slide containing sections with distilled water or buffer from a wash
bottle. Place slide at room temperature in a buffer bath for 5 minutes to rehydrate
sections.
2. Gently remove excess liquid from around the specimen. Avoid touching the tissue
directly
3. Apply 4-6 drops of normal serum, (normal serum from the host of the secondary
antibody) diluted 1:5–1:30 (final concentration. 3%–20%). Incubate for 20–30
minutes at 37 0C.
4. Tilt off serum and wipe away excess. Do not rinse.
5. Perform any antigen retrieval if necessary.
6. Apply 25-50 μL of rabbit (mouse) primary antibody diluted
appropriately per tissue section. Antibody should cover
sections completely. Incubate for desired time (see above for
suggested parameters and temperatures).
• If optimal antibody dilution is unknown, perform a series of
antibody dilutions in the range of 1:20–1:1,000 to obtain
initial results.
Note: Antibody diluent is often very important for consistent
reactivity. Simple solutions are easier to troubleshoot than
complex ones, thus antibodies diluted only with simple
buffers (Tris buffer saline (TBS) or PBS) are usually
recommended.
5.6 Methods of antibody detection
– Enzyme mediated;
– Fluorescence;
– Signal amplification.
• Two of the most commonly used detection methods are
fluorescence and colorimetric (enzyme mediated) detection.
• With the advent of electron microscopy, detection of antigens
by antibodies that contain large gold particles is often used
and these may also be visualized at the light microscopic level
as well, but their use is quite rare today, outside of electron
microscopy.
• Described below are the common antibody detection
methods for light microscopy.
 5.6.1 Enzyme-Mediated Detection
• When choosing a substrate for conversion by an enzyme, one
should select a substrate, which yields a precipitating product.
• Examples of commonly used substrates are listed below

Comments: 4-chloro-1-naphthol (CN) precipitates as a blue end product. Since CN is

soluble in alcohol and other organic solvents, the slides must not be dehydrated, exposed to alcoholic counter stains, or cover slipped
with mounting media containing organic solvents. Unlike DAB, CN tends to diffuse from the site of precipitation, thus it is not usually
recommended for Immunohistochemistry but can be used for Western blotting.
Comments: Napthol AS acts as the substrate for alkaline phosphatase, and the
Fast Red chromogen precipitates at the enzymatic sites producing a vibrant
red/pink color. Precipitate is soluble in alcohol, thus aqueous counter stain and
mounting medium should be used.
5.6.2 Fluorescence
• A molecule that fluoresces can be attached to the antibody
for detection using UV light.
• Examples are Fluorescein, Rhodamine, Texas Red®, Cy3 and
Cy5. In selecting fluorescence, one is limited by the available
microscope filter sets. Most filter sets are best matched with
rhodamine or fluorescein. Texas Red® may also be used with a
rhodamine filter set.
• Many mounting media contain “anti-fading” solutions, such as
DABCO, which will prolong the viewing time of the sample.
Chemicon offers a variety of fluorescence mounting fluids and
counterstain solutions including our basic fluorescent
mounting fluid and enhanced counterstaining fluid containing
nuclear stains such as DAPI and PI.
5.6.3 Signal Amplification
• Signal amplification techniques greatly enhance the sensitivity
of immunohistochemical and immunocytochemical methods.
• The signal amplification methods may be used in conjugation
with either of the above detection techniques.
• Signals may be amplified by using polyconjugated secondary
antibodies, or Avidin-Biotin interactions or other commercially
available amplifiers (i.e., tyramide catalyzed systems), which
increase the signal to antibody ratio.
• When signal amplification is used to amplify the specific
signal, however, one should be aware that non-specific signals
might also become amplified. Thorough washing and proper
antibody titration is especially important in this case.
5.7 Troubleshooting
• Trouble shooting procedures should be performed to check for:
– No staining;
– Weak staining;
– Background staining.
• When tissue staining has not given the expected results, the
experiment should be examined in a systematic way, wherein
only single experimental variables are altered at one time.
• Proper immunohistochemical troubleshooting requires one to
determine whether difficulties are related to specimen,
antibodies, technique, environment, or slide interpretation