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Immunohistochemistry
• Immunohistochemistry (IHC) is the process of selectively
imaging antigens (proteins) in cells of a tissue section by
exploiting the principle of antibodies binding specifically to
antigens in biological tissues.
IMMUNO HISTOCHEMISTRY • IHC takes its name from the roots "immuno", in reference to
Obed Ohene-Djan Atuahene, MLS antibodies used in the procedure, and "histo," meaning
tissue.
• Immunohistochemistry (IHC) combines histological,
immunological and biochemical techniques for the
identification of specific tissue components by means of a
specific antigen/antibody reaction tagged with a visible label
• Albert Coons conceptualized and first implemented the
procedure in 1941.

Principle
• The principle of immunohistochemistry is the
localization of antigens in tissue sections by the use
of labelled antibodies as specific reagents
• Antigen-antibody interactions that are visualized by
a marker such as fluorescent dye, enzyme,
radioactive element or colloidal gold.

Immunoslide Showing tubulin, actin and nucleus in green,

red and blue respectively.

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Immunostaining Patterns
• Nuclear: chromogenic expression targets the nuclear
content (chromatin).
• Membrane/Cytoplasmic: chromogenic expression is
localized around the membrane or within the cytoplasm.
Nuclear staining Membrane/Cytoplasmic staining
Disadvantages of IHC
1. Limited ability to quantitate protein content
2. Problems with antibody types, limited ability to detect
protein modifications.
3. Limited or lack of evidence based criteria.
4. Single or dual detection ability.
5. Variable scoring methods and reproducibility
6. No normalization methods
7. Limited throughput.
8. Limited capacity for clinical biomarker profiling (only
with tissue microarrays).
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Advantages of IHC
1. Protein location and distribution seen.
2. Detectable in small and large tissue biopsies and fixed
tissues.
3. Validation of other high-throughput studies (DNA
microarray)
Antigens
• Molecule that induces the formation of an antibody
and bears one or more antibody binding sites.
• These are highly specific topographical regions
composed of a small number of amino acids or
monosaccharide units, being known as antigenic
determinant groups or epitopes.
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Antigens
• The outer surfaces of antigens are covered by
unique 3-dimensional protein structures known as
epitopes.
• They are immunogenic - the ability to induce
antibody formation; and specifically reactive, which
means that the antigen can react “with the antibody
it caused to be produced.
Antibody
• Antibodies belong to the class of serum proteins
known as immunoglobulins.
• The terms antibody and immunoglobulin are often
used interchangeably. They are found in blood and
tissue fluids, as well as many secretions.
• The basic unit of each antibody is a monomer. An
antibody can be monomeric, dimeric, trimeric,
tetrameric, or pentameric.
• The monomer is composed of two heavy and two
light chains.
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Antibody
• There are five types of antibodies found in the blood of
higher vertebrates: IgA, IgD, IgE, IgG, and IgM.
• They are named for their heavy chains
• IgG has heavy chains of the gamma type.
• IgA, alpha heavy chains
• IgD, delta heavy chains
• IgE, epsilon heavy chains
• IgM mu heavy chains
• IgG is the commonest and the most frequently used
antibody for immunohistochemistry.
• A primary antibody for immunoperoxidase staining that
is "specific for gamma chains“ will localize the heavy
chain of an IgG molecule.
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Antibody
• There are only two types of light chains common to
all five groups: kappa and lambda.
• An IgG molecule has two identical light chains. Ie.
either two kappa chains or two lambda chains
• A single antibody can never have both kappa and
lambda chains.

Antibody production
• A source for the antigen such as serum , urine or
tissue is subjected to a combination of procedures
including precipitation, centrifugation, dialysis…
• Other techniques like chromatography and
electrophoresis to obtain a highly purified antigen.
• The antigen is then injected into an animal, not
same species.
• Antibody production begins within twenty minutes
after injection, although a measureable quantity of
antibody cannot be detected for 5-10 days.
• Bossting may be required

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MONOCLONAL POLYCLONAL
Antibody types Mouse or rabbit hybridoma Many different species (mostly rabbits)

• Polyclonal antibodies
• made by injecting animals with the protein of interest, or
a peptide fragment and, after a secondary immune
response is stimulated, isolating antibodies from whole
serum.
• Polyclonal antibodies are a heterogeneous mix of
antibodies that recognize several epitopes.
• Monoclonal antibodies
• made by injecting the animal and then taking a specific
sample of immune tissue, isolating a parent cell, and
using the resulting pure cell line to create antibodies.
• This causes the antibodies to show specificity for a single
epitope.
Tends to be cleaner and highly specific
More likely to get false- negative results if More likely to have success in an unknown
target epitope is damaged or altered
Expensive to produce
Training is required for the technology
used
Time scale is long for hybridomas
Recognizes only one epitope on an
antigen
Can produce large amount of specific
antibodies
unlimited supply
Tends to have more non-specific reactivity and
greater potential for false positive staining
application
Inexpensive
Skills required are low
Time scale is short
Recognizes multiple epitopes on ant one
antigen
Produces large amounts of non specific
antibodies with batch-to-batch variability
limited supply

Conjugation/ labelling Conjugation/ labelling

• Antibodies are not visible with standard microscopy Common labels
and must be labelled in a manner that does not • Flurochromes (fluorescein, rhodamine)
interfere with their binding specificity.
• Enzymes (peroxidase, alkaline phosphatase)
• Conjugation is the process of chemically linking
some type of marker onto an antibody molecule • Electron scattering compounds for use in electron
microscopy (ferritin, colloidal gold).
• A wide variety of conjugates are available for use in
various direct and indirect immunohistological stains

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Antibody detection IHC Methods

• For immunohistochemical detection strategies, • Direct
antibodies are classified as primary or secondary • Indirect
reagents.
• PAP
• Primary antibodies are raised against an antigen of
interest and are typically unconjugated (unlabeled), while
secondary antibodies are raised against immunoglobulins
of the primary antibody species.
• The secondary antibody is usually conjugated to a linker
molecule, such as biotin, that then recruits reporter
molecules, or the secondary antibody itself is directly
bound to the reporter molecule.

IHC Methods Direct Method

Direct
• The primary antibody is conjugated directly to the
label. The conjugate may be either a fluorochrome
or an enzyme.
• The labeled antibody reacts directly with the antigen in
the histological or cytological preparation.
• Quick and Easy but provides little signal amplification
and low on sensitivity.
• It is mainly confined to the demonstration of
immunoglobulin and complement in frozen sections of
skin and renal biopsies.
• Low levels of antigen present in certain tumors may not
be demonstrated by this technique

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IHC Methods Indirect Method

Indirect
• A labeled secondary antibody directed against the
immunoglobulin of the animal species in which the primary
antibody has been raised visualizes an unlabeled primary
antibody.
• Horseradish peroxidase labeling is most commonly used,
together with an appropriate chromogen substrate.
• More sensitive because multiple secondary antibodies may
react with different antigenic sites on the primary antibody,
thereby increasing the signal amplification.
• Same labelled secondary antibody can be used with a
variety of primary antibodies raised from the same animal
species.

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Avidin biotin-complex (ABC)
• Require a 2-step process of detection involving
biotinylated secondary antibody followed by
exposure to an avidin-peroxidase complex prior to
chromogenic detection.
Protocol
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IHC Methods
• READ
• Polymer chain two step indirect technique
• Unlabeled antibody enzyme complex techniques (PAP
and APAP)
• Immunogold silver staining technique (IGSS)
• Stretavidin-biotin techniques
• Hapten labelling technique
Step Effect on IHC
Biopsy Depending on the appropriate tissue type, tissue
samples can be obtained in different ways such as
punch/core biopsy, excisional/incisional etc.
Tissue degradation begins at the time of sample
removal.
Fixation The sample should be fixed as soon as possible after
sampling, ideally within less than an hour. The chemical
fixation crosslink proteins in the sample thereby
stopping the degradation process.
Embedding After fixation, the sample is embedded in paraffin for
long-term storage and to enable sectioning for
subsequent staining. Once embedded in paraffin,
samples can be stored (almost) indefinitely.
Sectioning Formalin-fixed, paraffin-embedded tissues are sectioned
and Mounting into thin slices (4-5 μm) with a microtome.
The sections are then mounted onto adhesive-coated
glass slides.
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Antigen Due to the fixation process, an antigen retrieval
Retrieval treatment is applied to unmask the epitopes, either by
heat (heat-induced epitope retrieval; HIER) or
enzymatic degradation (proteolytic-induced epitope
retrieval; PIER).
Primary The specificity and sensitivity of the antibody affect the
Antibody staining result.
Visualization The antigen/antibody complex signal is amplified and
visualized using a detection system. The strength of
amplification of the reaction affects the staining result
(intensity).
Interpretation The staining pattern is assessed by a pathologist in
context with other biomarkers, controls and other tests
(e.g. H&E, special stains.
Sample Preparation
• Specimens are typically sliced at a range of 3 µm-5
μm
• The slices are then mounted on slides, dehydrated
using alcohol washes of increasing concentrations
(e.g., 50%, 75%, 90%, 95%, 100%), and cleared
using a detergent like xylene before being imaged
under a microscope.
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Sample Preparation
• Critical to maintain cell morphology, tissue
architecture and the antigenicity of target epitopes.
• This requires proper tissue collection, fixation and
sectioning
• The tissue may then be sliced or used whole,
dependent upon the purpose of the experiment or
the tissue itself.
• Before sectioning, the tissue sample may be
embedded in a medium, like paraffin wax or
cryomedia.
Sample Preparation
• Depending on the method of fixation and tissue
preservation, the sample may require additional
steps to make the epitopes available for antibody
binding, including deparaffinization and antigen
retrieval.
• For formalin-fixed paraffin-embedded tissues,
antigen-retrieval is often necessary, and involves
pre-treating the sections with heat or protease.
• These steps may make the difference between the
target antigens staining or not staining.
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Fixation
Goals of fixation
• Prevent autolysis by rapidly terminating
enzymatic/metabolic activities
• Prevent bacterial decomposition
• Preserve tissue structure while stabilization and
hardening the tissue for processing
Slide Storage
• Freshly cut slides - For best results
• Slides may lose antigenic potential over time in
storage.
• If slides must be stored, do so unbaked at 4°C.
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Slide Preparation
IHC-P: Paraffin-embedded Cell Pellets and Tissue
• Prior to immunostaining, harvested and fixed in
10% neutral buffered formalin (NBF) to preserve cell
morphology and target epitopes.
IHC-F: Frozen Tissue
• Frozen tissue should be stored at -80°C
• When ready to stain, equilibrate tissue at -20°C for
15 minutes before attempting to section. Section the
tissue to a 6-8 μm thickness using a microtome
Deparaffinization/ rehydration
• Paraffin wax must be completely removed- for
staining
• This is done through a series of sequential
xylene/ethanol/water washes that remove the wax
and rehydrate the tissue for subsequent antibody
binding.
• Insufficient paraffin removal can lead to spotty,
uneven background staining.
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Visualising IHC Reactions IHC reporters (Labels)

• This can be accomplished in a number of ways. • Enzyme Labels
• In the most common instance, an antibody is conjugated • Fluorescent Labels
to an enzyme, such as peroxidase, that can catalyse a
colour-producing reaction (see immunoperoxidase • Radiolabels
staining).
• Alternatively, the antibody can also be tagged to a
fluorophore, such as fluorescein or rhodamine (see
immunofluorescence).

Enzyme Labels Enzyme Labels

• Enzymes are the most widely used labels in
immunohistochemistry, and incubation with a
chromogen using a standard histochemical method
produces a stable, colored reaction end product
suitable for the light microscope.
• The variety of enzymes and chromogens available
allow the user a choice of color for the reaction end
product
• Horseradish peroxidase is commonly used as an
antibody label for several reasons:
• Its small size does not hinder the binding of antibodies to
adjacent sites.
• The enzyme is easily obtainable in a highly purified form
and therefore the chance of contamination is minimized.
• It is a stable enzyme and remains unchanged during
manufacture, storage and application.
• Any endogenous activity is easily quenched.

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Enzyme Labels
• HRP in combination with the most favored
chromogen, 3,3α-diaminobenzidine
tetrahydrochloride (DAB), yields a crisp, insoluble,
stable, dark brown reaction end product.
• Reaction with DAB can be enhanced using nickel,
producing a deep purple/black staining.
• DAB is classed as a hazardous chemical and has
been reported to be a potential carcinogen
Enzyme Labels
Calf intestinal alkaline phosphatase
• Alternative enzyme tracer to horseradish
peroxidase, particularly in alkaline phosphatase-
anti-alkaline phosphatase (APAAP) method.
• Fast Red TR used with naphthol AS-MX phosphate
sodium salt gives a bright red reaction end product
which is soluble in alcohol.
• New fuchsin has been reported as giving a
permanent insoluble red product when mounted in
resinous mountant.
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Enzyme Labels
• Other chromogens used and colour rxn:
• 3-amino-9-ethylcarbazole - red
• 4-chloro-1-naphthol - blue
• Hanker-Yates reagent - dark blue
• α-naphthol pyronin - red-purple
• 2,2’ -azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS)
• Commercial kits include Vector Red, Vector Blue, Vector VIP
(purple) and 5-bromo-4-chloro-3-indolyl-phosphate
(BCIP/NBT) (blue/violet).
• NB
• It should be noted that some of these chromogens produce reaction
products which are soluble in alcohol and xylene, and therefore the
sections requireaqueous mounting.
Enzyme Labels
• Other labels such as glucose oxidase with nitroblue
tetrazolium, bacterial derived β-d-galactosidase,
colloidal gold and silver metals have been
developed but not suitable for routine use in the
diagnostic laboratory.
• Colloidal gold has a much wider use in the electron
microscope field.
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Fluorescent labels
• Fluorochromes are fluorescent labels which when
conjugated to the antibody absorb ultraviolet or
visible light of a particular wavelength to reach an
unstable excited state as its electrons gain energy.
• The fluorochrome subsequently emits light of a
different, usually longer, wavelength to that of the
excitation light as the electrons return to their
ground state.
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Fluorescent labels
• Different serum proteins bind differently to
fluorochromes.
• Immunoglobulins, in particular, have less affinity for
fluorescein isothiocyanate (FITC) than other more
‘negatively charged’ proteins such as albumin and
β-proteins.
• Fluorescent reporters traditionally include FITC,
Tetramethylrhodamine (TRITC) and
Aminomethylcoumarin (AMCA), while commercial
derivatives, include the Alexa Fluors and Dylight
Fluors,.
Fluorescent labels
• Advantages
• Hi-resolution, easy to double/triple label
• Better sub cellular detail
• Can be used with 3D microscopy/live imaging
• Disadvantages
• Background auto fluorescence
• Cost
• Lack of surrounding tissue/cellular detail
• Not permanent
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Comparing immune slides showing intense autofluors
(background staining) with treated slide that reduces
autofluorescence
Quantitive analysis in IHC
• For chromogenic and fluorescent detection
methods, densitometric analysis of the signal can
provide semi- and fully quantitative data,
respectively, to correlate the level of reporter signal
to the level of protein expression or localization.
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Radiolabels
• The use of radioisotopes as tracers requires
autoradiographic facilities, and developed from the
need for quantitation in IHC.
• Techniques involving the use of radioisotopes are
not used in the routine diagnostic laboratory.
Antigen retrieval
• Antigens can be masked by the chemical
processes involved in formalin fixation and paraffin
processing
• This reduces visualization and lead to mis-diagnosis
• Frozen sections were commonly used to bypass the
problem of “over fixation” but gives poor
morphology
• Unmasking of these antigens is required before
immunostaining to increase accessibility to the
antigens.
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Antigen retrieval
Techniques
• Proteolytic enzyme digestion
• Microwave oven radiation
• Combined microwave oven radiation and proteolytic
enzyme digestion
• Pressure cooker heating
• Decloaker heating
• Pressure cooker inside a microwave oven
• Autoclave heating
• Water bath heating
• Steamer heating
Proteolytic enzyme digestion
• Enzymatic digestion of tissue sections for epitope
unmasking is typically accomplished through
incubation in a solution of protease.
• All are effective, but they may result in increased
background staining because the cleavage is
nonspecific and some Ags might be negatively
affected.
• For those antibodies that require enzymatic retrieval
rather than heat methods, the recommended
enzyme and digesting conditions will be clearly
indicated on the product datasheet.
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Antigen retrieval
Techniques
• pressure cooking and autoclaving are considered to be
more effective than microwaving, due to the more
uniform heating and higher temperatures
• Pathways to achieving success here may include
• the breaking of cross-linkagesblock antibody access to target
epitopes
• the extraction of diffusible blocking proteins,
• the precipitation of proteins,
• the hydrolysis of Schiff's bases,
• calcium chelation,
• paraffin removal and the rehydration of tissue.
Proteolytic enzyme digestion
• Enzymes used include trypsin, proteinase K,
pronase, ficin and pepsin.
• The effect of protease induced epitope retrieval
(PIER) depends on the concentration and type of
enzyme, incubation parameters (time, temperature,
and pH), and the duration of fixation.
• The enzyme digestion time is inversely related to
the fixation time.
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Heat mediated antigen retrieval techniques
• Revolutionary method
• Mechanism involved in HIER is unknown, but final
effect is the reversion of conformational changes
produced during fixation.
• Heating can unmask epitopes by hydrolysis of
methylene crosslinks
Heat mediated antigen retrieval techniques
• Another possible theory was described by Morgan
JM et al. (1997), who postulated that large calcium
coordination complexes formed during formalin
fixation prevent antibodies from combining with
epitopes on tissue-bound antigens.
• High temperature weakens or breaks some of the
calcium coordinate bonds, but the effect is
reversible on cooling, because the calcium complex
remains in its original position.
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Heat mediated antigen retrieval techniques
• It also acts by other less known mechanisms
because it enhances immunostaining of tissue fixed
in ethanol, which does not produce cross-links.
• Other hypotheses proposed are
• extraction of diffusible blocking proteins,
• precipitation of proteins,
• rehydration of the tissue section allowing better
penetration of Ab, and
• heat mobilization of trace paraffin.
Heat mediated antigen retrieval techniques
Various types of equipment may be used
• including a de-cloaker (commercial pressure cooker
with electronic controls for temperature and time),
vegetable steamer, microwave oven, or pressure
cooker.
• The advantage of a de-cloaker over other heating
devices is that the boiling temperature is not
affected by the atmospheric pressure, which varies
depending on the altitude.
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Heat mediated antigen retrieval techniques
• The relationship between temperature and exposure
time is inverse: the higher the temperature, the shorter
the time needed to achieve beneficial results.
• pH of the retrieval solution is important. Some
antibodies bind well regardless of retrieval
solution pH, whereas others bind weakly at neutral pH,
but strongly at very low or high pH.
• Common buffers used in heat-induced AR are citrate,
Tris-HCl, and EDTA (ethylene-diaminetetraacetic acid).
• A low pH buffer (acetate, pH 1.0–2.0) appears
especially useful for nuclear antigens.
Heat mediated antigen retrieval techniques
Microwave antigen retrieval:-
• The actual heating time will depend on:
• Wattage of the oven.
• Choice of antigen retrieval buffer.
• Volume of buffer being used.
• Fixation of the tissues under investigation, in terms of
fixative used and duration of fixation.
• Thickness of tissue sections. 3μm sections require less
antigen retrieval than 5μm sections.
• Antigen to be demonstrated
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Heat mediated antigen retrieval techniques
Microwave antigen retrieval:-
• The method improves the demonstration of well
established antibodies such as CD45 and CD20
and enables the demonstration of a wide range of
new antibodies, such as CD8 and p53.
• Most domestic microwave ovens are suitable for
antigen retrieval and operate at 2.45GHz
corresponding to a wavelength in vacuum of
12.2cm.
Heat mediated antigen retrieval techniques
Pressure cooker antigen retrieval:-
• The pressure cooker could be used as an
alternative to the microwave oven.
• More uniform than microwave heating methods.
• A pressure cooker at 15psi (103kPa) reaches a
temperature that appears to be a major advantage
when unmasking nuclear antigens such as bcl-6,
p53, p21 etc.
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Heat mediated antigen retrieval techniques Heat mediated antigen retrieval techniques

• It is preferable to use a stainless steel domestic Steamer:-

pressure cooker, because aluminium pressure
cookers are susceptible to corrosion from some of
the antigen retrieval buffers.
• The pressure cooker should have a capacity of 4-5
litres.
• Steam heating appears to be less efficient than
either microwave heating or pressure cooking.
• Times in excess of 40 minutes are sometimes
required, but the method does have the advantage
in being less damaging to tissues than the other
heating methods.

Heat mediated antigen retrieval techniques

Water bath:- Autoclave:-

• A water bath set at 90°C is adequate for antigen • This method offers an alternative form of heat
retrieval.
mediated antigen retrieval, producing good results
• The technique has the advantage of being gentler on for nuclear antigens such as MIB1, p21 and p53
the tissue sections because the temperature is set
below boiling point.
• By using a lower temperature than other heating
methods the antigen retrieval buffer does not evaporate
and expensive commercial antigen retrieval solutions
can be safely reused.
• The method has the disadvantage in that the antigen
retrieval times are increased compared to other
methods.

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Reducing background/ non-specific
immuno-staining
• Although antibodies show preferential avidity for
specific epitopes, they may partially or weakly bind
to sites on nonspecific proteins (also called reactive
sites) that are similar to the actual binding sites on
the target antigen.
• Depending on the tissue type and the method of
antigen detection, endogenous biotin or enzymes
may need to be blocked or quenched, respectively,
prior to antibody staining.
Reducing background/ non-specific
immuno-staining
• Background staining may be specific or non specific
due to the apparent affinity of certain tissue
components.
• Tissues that give background staining include
collagen and other connective tissues, epithelium
and adipocytes.
• Hydrophobic bonding can be minimized by the
addition of a blocking protein, by the addition of a
detergent such as Triton X or the addition of a high
salt concentration, 2.5% NaCl to the buffer.
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Reducing background/ non-specific
immuno-staining
• A great amount of non-specific binding causes high
background staining which will mask the detection
of the target antigen producing inaccurate results.
• The major causes of background staining in IHC are
hydrophobic and ionic interactions and endogenous
enzyme activity.
Reducing Background staining
• To reduce background/non-specific staining,
samples are incubated with a buffer that blocks the
reactive sites to which the primary or secondary
antibodies may otherwise bind.
• Routinely, blocking the samples in buffered saline
plus 5% normal goat serum (NGS) for 1 hour at
room Temperature reduces background staining.
• Common blocking buffers include normal serum,
non-fat dry milk, BSA, or gelatin.
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Reducing Background staining
• Other Methods
• dilution of the primary or secondary antibodies,
• changing the time or temperature of incubation,
• using a different detection system or different
primary antibody.
• Quality control
• A tissue known to express the antigen as a positive
control and negative controls of tissue known not to
express the antigen should be used to validate the
results during visualization.
Blocking endogenous enzymes
• When using the hydrogen peroxide-methanol
method, care should be taken with certain antigens,
notably CD4
• Over incubation in the blocking solution or high
concentrations of hydrogen peroxide can significantly
diminish staining on formalin fixed, paraffin wax
embedded tissue.
• Performing the peroxidase block after the binding of
the primary antibody to the tissue antigen is
recommended for antibodies such as CD4
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Blocking endogenous enzymes
• Peroxidase and substances giving a pseudo-
peroxidase
reaction are present in some normal and neoplastic
tissues.
• These may produced an exaggerated colour
reaction in final stages and may lead to wrong
diagnosis.
• The most frequently used method is pre-incubation
of the sections in absolute methanol containing
hydrogen peroxide.
Blocking endogenous enzymes
• Alkaline phosphatases within the human body can
be blocked by using a 1mM concentration of
levamisole in the final incubating medium.
• Levamisole selectively inhibits certain types of
alkaline phosphatase, but not intestinal or placental,
when used at a concentration of 1 mM.
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Blocking endogenous enzymes
• Twenty percent glacial acetic acid inhibits all types
of alkaline phosphatase thus, a better blocker of
endogenous alkaline phosphatase.
• Another method includes the usage of one
chromogen and then, following IHC staining, the
enzyme tracer is localized by an alternate
chromogen which yields a reaction end-product in a
contrasting colour.
Additional notes on reducing high
background staining
• Blocking  may be improved by simply draining the
excess buffer instead of washing the tissue sample
prior to the addition of antibodies.
• Use a monoclonal primary antibody instead of a
polyclonal to reduce cross-reactivity.
• Use cross-adsorbed polyclonal antibodies to reduce
cross-reactivity.
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Additional notes on reducing high
background staining
• Carefully prepare tissue sample. Damage to the
tissue can cause diffuse staining.
• Prepare thinner sections if penetration of the
detection reagents is insufficient.
• Optimize fixation. Each tissue antigen will react
differently with different fixatives. Optimize the pH,
incubation time and temperature.
Additional notes on reducing high
background staining
• Affinity-purify the antibody preparation on an
immobilized antigen column. Many primary
antibodies and almost all secondary antibodies are
purified in this manner by their suppliers.
• Decrease the concentrations of the primary and/or
secondary antibodies to reduce nonspecific binding.
• Decrease the incubation times with the primary and
secondary antibodies to reduce nonspecific binding.
• Choose an improved substrate that will produce a
higher signal-to-noise ratio for the system such as
metal-enhanced DAB rather than DAB, or kits
based on TSA technology.
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Counterstains
• After immunohistochemical staining of the target
antigen, a second stain is often applied to provide
contrast that helps the primary stain stand out.
• Many of these stains show specificity for specific
classes of biomolecules, while others will stain the
whole cell.
• Both chromogenic and fluorescent dyes are
available for IHC and include: hematoxylin, Hoechst
stain and 4′,6-diamidino-2-phenylindole (DAPI).
Controls
Controls validate immunohistochemical results
• Positive controls:
• It is defined as tissue that is known to contain the antigen of
interest detected by identical IHC methods to those used in
diagnostic cases.
• Positive tissue controls must be fixed and staine in the same
way as the diagnostic case tissue for every antibody and
procedure used.
• Positive elements within test sections, e.g. normal reactive
lymphocytes when staining with an antibody to the leukocyte
common antigen to identify a suspected lymphoma, are the
best form of positive control.
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Mounting and examination
• Aqueous and non aqueous (permanent) mounting
media are available.
• The mounting media depends on the chromogen used
during the detection step and its solubility in organic
solvents or water.
• Water insoluble Chromogens should not be used with
non-aqueous mounting media and water soluble
chromogens should not be used with aqueous media.
• Nonaqueous mounting media is not compatible with
water; therefore, the samples must be first dehydrated
with a series of ethanol and xylene washes.
Controls
Negative controls:
• Negative tissue control is defined as tissue that is known not to
contain the antigen of interest.
• At least one ancillary test (e.g. PCR, virus isolation) performed on the
tissues/organ systems of the same animal should be used to rule out the
presence of the antigen of interest.
• This also involves the omission of the primary antibody from the staining
schedule or the replacement of the specific primary antibody by an
immunoglobulin which is directed against
an unrelated antigen.
• Commonly, the primary antibody is replaced by antibody diluent, same
species non-immune immunoglobulin of the same dilution and
immunoglobulin concentration, an irrelevant antibody or buffer.
• These methods will assess the degree of cross-reactivity of the primary
antibody, and the degree of nonspecific binding by the labeling
(secondary) antibody and detection system.
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Controls
Internal positive tissue controls:
• Internal positive tissue controls are present in
diagnostic case tissues.
• An example is the detection of smooth muscle
markers or vimentin in normal blood vessels. The
presence of positive staining in these areas
indicates appropriate immunoreactivity.
• With this type of control, there is no fixation
variable between the control tissue and the
diagnostic case tissue.
Immunohistochemistry of formalin-fixed paraffin-embedded (FFPE) cancer tissue. Analysis was
performed to compare Connexin 43 membrane staining in FFPE sections of human lung
adenocarcinoma (right) compared to a negative control without primary antibody (left)
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Hematoxylin and eosin (HE) staining and immunohistochemistry analyses of bone marrow (BM),
spleen, and lymph nodes (LNs) from autopsy. HE staining of BM showed hypercellularity, and
increased blasts. These blasts expressed myeloperoxidase (MPO), whereas the population of
lymphocytes was small and there was almost no staining for encoded small nuclear RNAs (EBERs)
in the BM. The spleen tissue also showed diffuse infiltration of myeloblasts, and rare EBER-positive
lymphocytes. In contrast, leukemic cells and EBER-positive lymphocytes were seen in the LNs.
Molecular Marker Description
• Epithelial marker: keratins
• General mesenchymal markers: vimentin
• Muscle markers: desmin, actins, myoglobins, myogenin
• Neural markers: S100, GFAP, neurofilaments, CD57
• Endothelial markers: CD31, CD34, factor VIII related
antigen
• Lymphoid markers: к and λ, CD3, CD15, CD20,
CD30,CD45, CD68, CD79a,ALK-1 and TdT
• Neuroendocrine markers: synatophysin, chromogranin
• Metastatic tumor markers: CK7, CK20 and villin
• Minor salivary gland tumor markers: S-100 protein,
actins
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Immunohistochemistry (IHC) vs. Immunocytochemistry (ICC) Immunohistochemistry (IHC) vs. Immunocytochemistry (ICC)

• IHC is performed on samples derived from tissues
that have been histologically processed into thin
sections and the staining process exploits enzymes
which catalyze the deposition of a colored staining
product at antigenic sites within the sample.
• ICC relies on the same enzyme reactions as IHC,
but it is performed on samples consisting of cells
grown in a monolayer or cells in suspension which
are deposited on a slide.
• Prior to IHC, tissues are removed from the patient
or animal and either frozen or chemically preserved
(fixed) and then embedded in paraffin wax.
• Sections as thin as 4 μm are sliced from frozen or
paraffin-embedded tissue and mounted onto slides
in preparation for antibody-mediated staining.
• This enables visualization of the localization of the
target antigens within cellular components while
maintaining the original architecture of the
surrounding tissue.

Immunohistochemistry (IHC) vs. Immunocytochemistry (ICC) Immunohistochemistry (IHC) vs. Immunocytochemistry (ICC)

• For ICC, most, if not all, of the extracellular matrix

and other stromal components are removed,
leaving only whole cells.
• Sample sources for ICC can be from any
suspension of cells, obtained from patients or
animals (e.g., blood smears, swabs and aspirates)
or cultured cells grown in monolayers (eg. sterile
glass coverslips).
• ICC is also synonymous with IF staining, especially
when we present results with cultured cells. The 2
abbreviations are often used interchangeably in this
context.

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Immunohistochemistry (IHC) vs. Immunocytochemistry (ICC)
• IHC and ICC differ in the amount of sample processing
required before antibody-mediated staining.
• ICC is associated with whole cells that have to be
permeabilized, either through a fixation procedure or a
separate permeabilization step, to facilitate antibody
penetration to the intracellular targets.
• Depending on the thickness of the section and the
method of fixation, IHC samples may not have to
undergo a separate permeabilization step.
• However, most formalin-fixed, paraffin-embedded (FFPE) IHC
sections must be further processed prior to antibody staining
to uncover latent epitopes on antigenic targets in a process
referred to as epitope or antigen retrieval.
IHC in Application
• IHC is typically applied to cases when the definitive
diagnosis cannot be established on the sole basis
of findings in H&E sections.
• In a routine surgical pathology service, the
maximum utility of IHC is in distinguishing
carcinoma from lymphoma and melanoma and also
in the work-up of hematolymphoid neoplasms.
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Applications
• Immunohistochemical staining is widely used in the
diagnosis of abnormal cells such as those found in
cancerous tumors.
• Specific molecular markers are characteristic of
particular cellular events such as proliferation or cell
death (apoptosis).
• Useful in monitoring cancer treatment
• Immunohistochemistry is also widely used in basic
research to understand the distribution and
localization of biomarkers and differentially
expressed proteins in different parts of a biological
tissue.
IHC in Application
The following are some of the conditions where IHC
staining has been proved to be immensely useful:-
• Identifying the presence of genetic mutations and
chromosomal translocations.
• To identify therapeutic targets for cancer.
• Diagnosing papillary and intraductal proliferations, low-
grade carcinomas, radial scars, sclerosing adenosis,
tubular carcinomas and lobular carcinomas of the breast.
• Diagnosis of malignant round cell tumors.
• In detecting metastatic carcinomas of unknown origin.
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IHC in Application Future Directions

Application of IHC in prognostic and predictive settings.
The prognostic markers include those that allow
• assessment of microscopic invasion of basal lamina,
• micro-metastasis to sentinel/regional nodes and bone
marrow,
• hormone receptors (ER/PR), angiogenesis, tumor
associated genes including p53, growth factor receptors
and anti-metastasis genes and
• markers that predict response to therapy such as
glycoprotein and c-erb-2.2,8
• genomic IHC for diagnosis,
• search for proteins for targeted therapy,
• methods to develop better monoclonal antibodies
with recombinant technology,
• ''technician free'' automation of the IHC procedures,
and ''pathologist free'' microscopic image analysis
• technology for interpretation of high throughput
results.

Bibliography and Further Reading

• Bancroft, J. D., & Gamble, M. (Eds.). (2008). Theory and practice of
histological techniques. Elsevier Health Sciences.
• Kabiraj, A., Gupta, J., Khaitan, T., & Bhattacharya, P. T. (2015). Principle
and techniques of Immunohistochemistry-a review. Int J Biol Med Res,
6(3), 5204-5210.
• Asiamah, E., (2018) Immunohistochemistry Basics Presentation
• ThermoFischer, IHC Troubleshooting Guide (Available Online)
https://goo.gl/BnmgDW
• Nelson, G. (2018) Cell Cytoskeleton (Available Online)
https://goo.gl/g4WJTV

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