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Immuno Histochemistry
Immuno Histochemistry
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.
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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 Antibody
• Antibodies belong to the class of serum proteins • There are five types of antibodies found in the blood of
higher vertebrates: IgA, IgD, IgE, IgG, and IgM.
known as immunoglobulins.
• They are named for their heavy chains
• The terms antibody and immunoglobulin are often • IgG has heavy chains of the gamma type.
used interchangeably. They are found in blood and • IgA, alpha heavy chains
tissue fluids, as well as many secretions. • IgD, delta heavy chains
• The basic unit of each antibody is a monomer. An • IgE, epsilon heavy chains
antibody can be monomeric, dimeric, trimeric, • IgM mu heavy chains
tetrameric, or pentameric. • IgG is the commonest and the most frequently used
antibody for immunohistochemistry.
• The monomer is composed of two heavy and two • A primary antibody for immunoperoxidase staining that
light chains. 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 Tends to be cleaner and highly specific Tends to have more non-specific reactivity and
greater potential for false positive staining
• made by injecting animals with the protein of interest, or
a peptide fragment and, after a secondary immune More likely to get false- negative results if More likely to have success in an unknown
target epitope is damaged or altered application
response is stimulated, isolating antibodies from whole
serum. Expensive to produce Inexpensive
• Polyclonal antibodies are a heterogeneous mix of Training is required for the technology Skills required are low
antibodies that recognize several epitopes. used
Time scale is long for hybridomas Time scale is short
• Monoclonal antibodies
Recognizes only one epitope on an Recognizes multiple epitopes on ant one
• made by injecting the animal and then taking a specific antigen antigen
sample of immune tissue, isolating a parent cell, and Can produce large amount of specific Produces large amounts of non specific
using the resulting pure cell line to create antibodies. antibodies antibodies with batch-to-batch variability
• This causes the antibodies to show specificity for a single
epitope. unlimited supply limited supply
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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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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.
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Heat mediated antigen retrieval techniques Heat mediated antigen retrieval techniques
Heat mediated antigen retrieval techniques Heat mediated antigen retrieval techniques
• Another possible theory was described by Morgan Various types of equipment may be used
JM et al. (1997), who postulated that large calcium • including a de-cloaker (commercial pressure cooker
coordination complexes formed during formalin with electronic controls for temperature and time),
fixation prevent antibodies from combining with vegetable steamer, microwave oven, or pressure
epitopes on tissue-bound antigens. cooker.
• High temperature weakens or breaks some of the • The advantage of a de-cloaker over other heating
calcium coordinate bonds, but the effect is devices is that the boiling temperature is not
reversible on cooling, because the calcium complex affected by the atmospheric pressure, which varies
remains in its original position. depending on the altitude.
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Heat mediated antigen retrieval techniques Heat mediated antigen retrieval techniques
Heat mediated antigen retrieval techniques Heat mediated antigen retrieval techniques
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Heat mediated antigen retrieval techniques Heat mediated antigen retrieval techniques
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• Although antibodies show preferential avidity for • A great amount of non-specific binding causes high
specific epitopes, they may partially or weakly bind background staining which will mask the detection
to sites on nonspecific proteins (also called reactive of the target antigen producing inaccurate results.
sites) that are similar to the actual binding sites on • The major causes of background staining in IHC are
the target antigen. hydrophobic and ionic interactions and endogenous
• Depending on the tissue type and the method of enzyme activity.
antigen detection, endogenous biotin or enzymes
may need to be blocked or quenched, respectively,
prior to antibody staining.
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Controls Controls
Controls validate immunohistochemical results Negative controls:
• Negative tissue control is defined as tissue that is known not to
• Positive controls: contain the antigen of interest.
• It is defined as tissue that is known to contain the antigen of • At least one ancillary test (e.g. PCR, virus isolation) performed on the
interest detected by identical IHC methods to those used in tissues/organ systems of the same animal should be used to rule out the
presence of the antigen of interest.
diagnostic cases.
• This also involves the omission of the primary antibody from the staining
• Positive tissue controls must be fixed and staine in the same schedule or the replacement of the specific primary antibody by an
way as the diagnostic case tissue for every antibody and immunoglobulin which is directed against
procedure used. an unrelated antigen.
• Positive elements within test sections, e.g. normal reactive • Commonly, the primary antibody is replaced by antibody diluent, same
species non-immune immunoglobulin of the same dilution and
lymphocytes when staining with an antibody to the leukocyte immunoglobulin concentration, an irrelevant antibody or buffer.
common antigen to identify a suspected lymphoma, are the
• These methods will assess the degree of cross-reactivity of the primary
best form of positive control. 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
Hematoxylin and eosin (HE) staining and immunohistochemistry analyses of bone marrow (BM),
variable between the control tissue and the 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
diagnostic case tissue. 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.
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Immunohistochemistry (IHC) vs. Immunocytochemistry (ICC) Immunohistochemistry (IHC) vs. Immunocytochemistry (ICC)
• IHC is performed on samples derived from tissues • Prior to IHC, tissues are removed from the patient
that have been histologically processed into thin or animal and either frozen or chemically preserved
sections and the staining process exploits enzymes (fixed) and then embedded in paraffin wax.
which catalyze the deposition of a colored staining • Sections as thin as 4 μm are sliced from frozen or
product at antigenic sites within the sample. paraffin-embedded tissue and mounted onto slides
• ICC relies on the same enzyme reactions as IHC, in preparation for antibody-mediated staining.
but it is performed on samples consisting of cells • This enables visualization of the localization of the
grown in a monolayer or cells in suspension which target antigens within cellular components while
are deposited on a slide. maintaining the original architecture of the
surrounding tissue.
Immunohistochemistry (IHC) vs. Immunocytochemistry (ICC) Immunohistochemistry (IHC) vs. Immunocytochemistry (ICC)
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