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DR KALPAJYOTI BHATTACHARJEE
• Production of primary reagents
• Immonohistochemistry methods
• Tissue fixation
• Unmasking of antigen sites
• Immunohistochemistry in practice
• current application
• Immunohistochemistry is as the name implies, a combination of two
disciplines – immunology and histology. The Immunohistochemistry
technique is used not only to determine if a tissue express or does not
express a particular antigen but also to determine the antigenic status of
particular cells within that tissue and microantomic location of the
• Immunohistochemistry uses antibodies to distinguish antigenic
difference between the cells
• This is a technique for identifying cellular or tissue constituents
(antigens) by means of antigen antibody interactions, the site of
antibody binding being identified either by direct labeling of the
antibody, or by use of a secondary labeling method.
• Immunohistochemistry- using tissue sections.
• Immunocytochemistry – cytological preparations
• An antigen is a 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
• The outer surfaces of antigens are covered by unique 3- dimensional
protein structures known as epitopes
• 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.
• There are five types of antibody found in the blood of higher vertebrates: IgA,
IgD, IgE, IgG, and IgM. IgG is the commonest and the most frequently used
antibody for immunohistochemistry.
• The associated antibody and antigen are held together by a
combination of hydrogen bonds, electrostatic interactions and
van der Waals’ forces.
• Affinity is the three-dimensional fit of the antibody to its specific antigen
and is a measure of the binding strength between the antigenic epitope and
its specific antibody-combining site.
• Avidity is a related property referring to the heterogeneity of the
antiserum which will contain various antibodies reacting with different
epitopes of the antigen molecule. Avidity therefore is the functional
combining strength of an antibody with its antigen.
• This refers to the characteristics of an antibody to bind selectively to a
single epitope on an antigen
• This refers to the relative amount of antigen that an
immunohistochemical technique is able to detect.
• The principle has existed since the 1930s.
• Started in 1941 when Albert H. Coons et al tagged fluorescein dye to
antibodies against pneumococci.
• Nakane and pierce et al. (1967) introduced enzyme peroxidase enabling IHC
staining to be viewed with light microscope.
• Colloidal gold label (Faulk andTaylor 1971) has also been discovered and used
to identify immunohistochemical reactions at both light and electron
• Mason and Sammons in 1978 introduced alkaline phosphatase.
• In 1979, Sternberger described the peroxidase-antiperoxidase (PAP) method.
Principle of Immunohistochemistry:
• Immunohistochemistry is a method for localizing specific antigen in
tissues or cells based on antigen antibody reaction.
• The site of antibody binding is identified either by tagging the antibody,
directly or indirectly with a visible label.
• Fluorescent dye, colloidal metal, hapten, radioactive marker.
Production of primary reagents
• It is the pivotal reagent common all IHC techniques.
• They impart specificty.
• Two types of antibodies are used:
• Polyclonal antibodies.
• Monoclonal antibodies.
• They are a heterogenous mixture of antibodies directed against various epitopes
of same antigen.
• Generated by different B-cell clones of the animals → immunochemically
• Produced by immunizing an animal with a purified specific molecule
(immunogen) bearing the antigen of interest.
• The animal will mount a humoral response to the immunogen and the
antibodies so produced can be harvested by bleeding the animal to obtain
• It is likely that the animal will produce numerous clones of activated plasma
cells (polyclonal). A polyclonal antiserum is therefore a mixture of antibodies
to different epitopes on the immunogen.
• They are a homogenous population of Ig directed against a single epitope.
• Generated by a single B-cell clone from one animal → immunochemically
• Developed by Kohler and Milstein in 1975.
• The method combines the ability of a plasma cell or transformed B
lymphocyte to produce a specific antibody with the in vitro immortality of a
neoplastic myeloma cell line..
• With the technique of cloning, this cell can be grown and multiplied in cell
culture or ascetic fluid, theoretically to unlimited numbers.
• The result is a constant, reliable supply of one pure antibody with known
Mouse or rabbit hybridoma Many different species (mostly rabbits)
Tends to be cleaner Tends to have more non-specific reactivity
More likely to get false- negative results if
target epitope is damaged or altered
More likely to have success in an unknown
Expensive to produce Inexpensive
Training is required for the technology
Skills required are low
Time scale is long for hybridomas Time scale is short
Recognizes only one epitope on an
Recognizes multiple epitopes on ant one
Can produce large amount of specific
Produces large amounts of non specific
1) 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
• Horseradish peroxidase is the most widely used enzyme, and in
combination with the most favored chromogen, i.e. 3,3α-
diaminobenzidene tetrahydrochloride (DAB).
• Horseradish peroxidase is commonly used as an antibody label for
Its small size does not hinder the binding of antibodies to adjacent sites.
Chance of contamination is minimized.
Endogenous activity is easily quenched.
• Calf intestinal alkaline phosphatase is the most widely used
alternative enzyme tracer to horseradish peroxidase, particularly since
the development of the alkaline phosphatase-anti-alkaline phosphatase
(APAAP) method in 1984 by Cordell et al.
• Bacterial-derived β-D-galactosidase has also been used as a tracer
2) Colloidal metal labels
• When used alone, colloidal gold conjugates appear pink when viewed
using the light microscope. An silver precipitation reaction can be used to
amplify the visibility of the gold conjugates.
3) Fluorescent labels
• 3,3α-diaminobenzidene tetrahydrochloride (DAB), it yields a crisp,
insoluble, stable, dark brown reaction end-product. Although DAB has been
reported to be a potential carcinogen, the risk is now thought to be low.
• 3-amino-9-ethylcarbazole -red
• 4-chloro-1-naphthol - blue;
• Hanker-Yates reagent - dark blue
• α-naphthol pyronin - red-purple
Traditional direct technique
• The primary antibody is conjugated directly to the label. The conjugate
may be either a fluorochrome (more commonly) or an enzyme. The
labeled antibody reacts directly with the antigen in the histological or
• Quick and easy to use.
• Provides little signal amplification
• Lacks the sensitivity.
New Direct technique
• Pluzek et al in 1993
• Commercial name- Enhanced Polymer One-step Staining (EPOS)
• A large number of primary antibody molecules and peroxidase enzymes
are attached to a dextran polymer ‘backbone’, hence increasing the signal
amplification and provide greater sensitivity.
Two-step indirect technique
• 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 technique because multiple secondary antibodies may
react with different antigenic sites on the primary antibody, thereby
increasing the signal amplification.
Polymer chain two-step indirect technique
• This technology uses an unconjugated primary antibody, followed by a
secondary antibody conjugated to an enzyme (horseradish peroxidase)
labeled polymer (dextran) chain.
• Conjugation of both anti-mouse and anti-rabbit secondary antibodies
enables the same reagent to be used for both monoclonal (rabbit and
mouse) and polyclonal (rabbit) primary antibodies.
Unlabeled Antibody Methods
PEROXIDASE ANTIPEROXIDASE METHOD / PAP
• Immune complex typically consists of 2 antibody molecules and 3 HRP
molecules in the configuration.
• The PAP reagent and the primary antibody must be from the same species,
whereas the bridge or linking antibody is derived from a second species and
has specificity against the primary antibody and the immunoglobulin
incorporated into the PAP complex.
ALKALINE PHOSPHATASE–ANTIALKALINE PHOSPHATASE
METHOD / APAAP
• Principle same as those described for the PAP method except that the PAP
complex is replaced with an APAAP complex.
The method has had three major
(1)staining of tissues with high levels of
(2)double immunostaining in conjunction
(3)staining of specific cell types that benefit
from the bright red color of alkaline
Immunogold silver staining technique (IGSS)
• Introduced by Faulk andTaylor (1971).
• In this method the gold particles are enhanced by the addition of metallic
silver layers to produce a metallic silver precipitate which overlays the
colloidal gold marker.
• silver lactate as the ion supplier
• hydroquinone as the reducing agent.
• Disadvantage: Formation of fine silver deposits in the background
• The biotin-avidin procedure exploits the high affinity binding between
biotin and avidin.
Biotin is linked chemically to the primary antibody,
Produces biotinylated conjugate that localizes to the sites of antigen
Avidin which is chemically conjugated to horseradish peroxidase, is added;
avidin binds tightly to the biotinylated antibody, thus
localizing the peroxidase moiety at the site of antigen in the tissue section.
1) Different batches of biotin and different batches of avidin have
differing affinities for one other → affects the sensitivity
2) Produces non- specific (false-positive) staining.
(Strept) avidin-biotin techniques
• The labeled streptavidin-biotin technique is the most widely used
methodology in diagnostic immunohistochemistry.
• 3 -step technique:
1. unconjugated primary antibody as the first layer,
2. followed by a biotinylated secondary antibody.
3. The third layer is either a complex of enzyme-labeled biotin and
streptavidin, or enzyme-labeled streptavidin
The enzyme can be either horseradish peroxidase or alkaline phosphatase,
used with a chromogen of choice
• Streptavidin has now largely replaced the use of avidin in
immunohistochemical detection techniques.
Biotinylated tyramide signal amplification
• Bobrow et al. first described the use of biotinylated tyramide to enhance
signal amplification, in 1989.
• The technique is based around the streptavidinbiotin technique.
• Advantage: Enables many antigens which had previously been unreactive
in formalin-fixed paraffin-embedded tissue to be demonstrated.
• Disadvantage: Excessive background staining
Application of the primary antibody
subsequent incubations in biotinylated secondary
horseradish peroxidase-labeled streptavidin
subsequent treatment with the biotinylated tyramide
free biotin radicals.
These reactive biotin molecules bind covalently to
proteins adjacent to the site of the reaction.
• A prerequisite for all routine histological and cytological investigations is
to ensure preservation of tissue architecture and cell morphology by
adequate and appropriate fixation.
• The fixative should preserve antigenic integrity and should limit
extraction, diffusion, or displacement of antigen during subsequent
• Fixation prevents the autolysis and necrosis of excised tissues, enhances
the refractive index of tissue constituents and increases the resistance of
cellular elements to tissue processing.
• Show good preservation of morphologic details after embedding in a
support medium (e.g., paraffin).
• Good fixation is the delicate balance between under-fixation and over-
• Ideal fixation is the balance between good morphology and good
• Prompt fixation is essential to achieve consistent results.
• Poor fixation or delay in fixation causes loss of antigenicity or diffusion of
antigens into the surrounding tissue.
• Fixative can cause changes in the steric configuration of proteins, which
may mask antigenic sites (epitopes) and adversely affect binding with
• It is well recognized that cross-linking fixatives (formaldehyde) alter the IHC
results for a significant number of antigens, whereas coagulant fixatives,
especially ethanol, have been reported to produce fewer changes, although
there remains some controversy.
• A robust and optimized fixation protocol is a critical step in an
immunohistochemistry protocol as an antigen that has been inappropriately
fixed may not be detected in downstream detection.
• The most popular choice of fixatives for routine histology are formalin
based, either as a 10% solution or with the addition of different chemical
• When formalin-based fixatives are used, intermolecular and intramolecular
cross-linkages are formed with certain structural proteins. These are
responsible for the masking of the tissue antigens.
• Methylene bridges forms between reactive sites on tissue proteins.
Advantages of formalin:
• good preservation of morphology, even after prolonged fixation
• economical chemical
• sterilizes tissue specimens in a more reliable way than precipitating
fixatives, particularly for viruses.
• Carbohydrate antigens are well preserved
• Cross-linking of protein in situ avoids leaching out of proteins that may
diffuse in water or alcohol.
• Many low–molecular-weight antigens (peptides) are well preserved in tissue
Unmasking of antigen sites
Manual methods for antigen unmasking include:
• Proteolytic enzyme digestion
• Microwave oven irradiation
• Combined microwave oven irradiation and proteolytic enzyme digestion
• Pressure cooker heating
• Decloaker heating
• Pressure cooker inside a microwave oven
• Autoclave heating
• Water bath heating
• Steamer heating
Before antigen unmasking pretreatments are employed, the sections are
dewaxed, rinsed in alcohol, and washed in water.
Proteolytic enzyme digestion
• Described by Huang et al. (1976), Curran and Gregory (1977), and
Mepham et al. (1979).
• The most popular enzymes employed today are trypsin and protease, but
other proteolytic enzymes such as chymotrypsin, pronase, proteinase K,
and pepsin may also be used.
• Principle- Digestion breaks down formalin cross-linking and hence the
antigenic sites for a number of antibodies are uncovered.
• Under-digestion results in too little staining, because the antigens are not
• Over-digestion can produce false positive staining, high background
levels, and tissue damage.
Heat-mediated antigen retrieval techniques
• Heat-based antigen retrieval methods have brought a great improvement in
the quality and reproducibility of immunohistochemistry.They have also
widened its use as an important diagnostic tool in histopathology.
Heavy metal salts (as described by Shi et al. 1991) act as a protein
precipitant, forming insoluble complexes with polypeptides, and
that protein precipitating fixatives display better preservation of
antigens than do cross-linking aldehyde fixatives
During formalin fixation intermolecular and methylene bridges and weak
Schiff bases form intramolecular cross-linkages, which may prevent it from
being recognized by a specific antibody.
Heat-mediated antigen retrieval removes the weaker Schiff bases but does
not affect the methylene bridges, so the resulting protein conformation is
intermediate between fixed and unfixed.
Morgan et al. (1997), who postulated that 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
Microwave antigen retrieval
• Shi et al. (1991) first established the use of microwave heating for antigen
• Gerdes et al. (1992) used microwave antigen retrieval with a non-toxic
citrate buffer at pH 6.0 .
• Cattoretti et al. (1993) established microwave oven heating as an alternative
to proteolytic enzyme digestion.
• Antigen retrieval solutions: 0.01 M citrate buffer at pH 6.0 and 0.1 mM EDTA
at pH 8.0
• Uneven heating and the production of hot spots
Pressure cooker antigen retrieval
• Norton et al. (1994) suggested the use of the pressure cooker as an
alternative to the microwave oven. Batch variation and production of hot
and cold spots in the microwave oven could be overcome.
• Pressure cooking is said to be more uniform than other heating methods.
• A pressure cooker at 15 psi (10.3 kPa) reaches a temperature of around
120°C at full pressure
• It is preferable to use a stainless steel domestic pressure cooker, because
aluminum pressure cookers are susceptible to corrosion from some of the
antigen retrieval buffers
• Steam heating appears to be less efficient than either microwave oven
heating or pressure cooking
• Advantage - less damaging to tissues than the other heating methods
• Kawai et al. (1994) demonstrated that a water bath set at 90°C was
adequate for antigen retrieval.
• Increasing the temperature to 95–98°C, antigen retrieval was improved and
the incubation times could be decreased.
Advantage - gentler on the tissue sections because the temperature is set
below boiling point.
• antigen retrieval buffer does not evaporate and
• expensive commercial antigen retrieval solutions can be safely reused
Disadvantage -antigen retrieval times are increased compared to other
Combined microwave antigen retrieval and trypsin digestion
• Infrequently used today;
• Brief proteolytic digestion can be carried out before or after microwave
Advantages of heat pretreatment
• Some antigens previously thought lost in routinely processed paraffin-
embedded sections are now recovered by heat pretreatment.
• Many antigens are retrieved by uniform heating times, regardless of
length of fixation.
Pitfalls of heat pretreatment
• Care should be taken not to allow the sections to dry after heating, as
this destroys antigenicity.
• The boiling of poorly fixed material also damages nuclear detail.
• Fibrous and fatty tissues tend to detach from the slide.
• Vectabond or APES-coated slides (3-Aminopropylenetriethoxysilane)
can be dipped in 10% formal saline for 1–2 minutes and air dried before
picking up the sections.
Commercial antigen retrieval solutions
• There are numerous commercial antigen retrieval solutions available.They
can be either specialized high pH solutions (recommended for certain
antibodies) or lower pH 6.0 for more general use.
• Citrate buffer pH 6.0
• EDTA buffer pH 8.5
• Pepsin reagent
• Tris-HCL buffer pH- 10
Immunohistochemistry in practice
• The choice of technique to suit the needs of particular types of work is
governed by some important factors.
• Although the use of frozen sections for diagnostic purposes is decreasing,
immunohistochemistry on frozen sections remains an important
Advantage: preserves enzyme and antigen function
• Poor morphology
• Limited prospective studies
• Storage of material difficult
• Cutting difficulty over paraffin sections
• Poor morphology → improved by ensuring the frozen sections are thoroughly
dried both before and after the sections are fixed in acetone.
• Acetone assists preservation of the antigen and related morphology and
also destroys most harmful infective agents.
• Acetone-fixed smears are often preferred by the immunohistochemist as
it allows a wide range of primary antibodies to be employed without
destroying the target epitopes
• In alcohol, consequently the number of antigens demonstrable may be
limited, although perhaps the morphology is superior.
Blocking endogenous enzymes
• If enzymes similar to those used as the antibody label are present in the
tissue, they may react with the substrate used to localize the tracer and
give rise to problems in interpretation.
• Inhibiting endogenous enzyme activity prior to staining can eliminate
• Tissues incubated with DAB substrate prior to primary antibody
incubation- if tissue turns brown- peroxidase present and blocking steps
• Incubation in absolute methanol containing 0.5% hydrogen
peroxide for 10 minutes at room temperature.
• Most endogenous alkaline phosphatase activity can be blocked by:
Adding levamisole in the final incubating medium.
Using 20% acetic acid can block intestinal alkaline phosphatase.
• Proteins blocked by: 10% normal serum
Blocking background staining
• The major causes of background staining in immunohistochemistry are
hydrophobic and ionic interactions and
endogenous enzyme activity.
• Non-specific uptake of antigen, particularly the high affinity of collagen and
reticulin for immunoglobulins, can cause high levels of background staining.
Hydrophobic interactions :
• Tissues that give background staining as a result of hydrophobic
interactions include collagen and other connective tissues, epithelium, and
• Addition of a blocking protein,
• Addition of a detergent such asTriton X ,
• Addition of a high salt concentration, 2.5% NaCl, to the buffer.
• Addiion of the blocking serum to the diluted primary antibody.
• Non-specific staining is most commonly produced because the primary
antibody is attracted nonimmunologically to highly charged groups
present on connective tissue elements.
• Prevention: Add an innocuous protein solution to the section before
applying the primary antibody.
• Traditionally, non-immune serum from the animal species in which the
second (bridging) antibody was raised is used as a blocking serum.
• Controls validate immunohistochemical results.
• It is essential that any method using immunohistochemistry principles
include controls to test for the specificity of the antibodies involved.
• Negative control. This involves either 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
• Positive control. Cells or tissues that are known to contain the specific Ag
Detects false negatives due to fixation and processing.
It is used to validate the protocol or procedure used
The final step in the process is counterstaining and mounting slides.
counterstains used are:Haematoxylin
Hoechst stain and
Hematoxylin is used as the nuclear counterstain for most routine
• Hoechst stains are part of a family of blue fluorescent dyes used
to stain DNA. There are three related Hoechst stains: Hoechst 33258,
Hoechst 33342, and Hoechst 34580.
• DAPI (4',6-diamidino-2-phenylindole) is a fluorescent stain. It is used
extensively in fluorescence microscopy. As DAPI can pass through an
intact cell membrane, it can be used to stain both live and fixed cells,
Limitations of Immunohistochemistry:
1. Experience: Experience is critical in standardizing the procedure
including the selection and proper dilutions of necessary reagents and
regular performance of all the appropriate controls. Interpretation too has
its foundation in experience.
2. Availability of antibodies: The paucity of antibody with high degree of
specificity for cellular and tissue antigens was serious limitation until
recently. This has been remedied in part by using hybridoma technique for
3. Antigen loss: The specificity of an antibody for particular antigen and its
ability to react with that antigen require the preservation of antigen
Current applications of immunohistochemistry
• Classification of Neoplasma
• Diagnosis of Malignancy
• Prognostic Markers
• Predicting response to treatment
• Detection of metastases
• Screening of inherited cancer syndromes
Non- Tumor Pathology
• Neurodegenerative diseases
• Brain trauma
• Muscle diseases