PRESENTED BY:DR KALPAJYOTI BHATTACHARJEE

IMMUNOHISTOCHEMISTRY METHODS

Contents

• Introduction •Definitions•History• Principle• Production of primary reagents• Technique• Immonohistochemistry methods• Tissue fixation •Unmasking of antigen sites• Immunohistochemistry in practice• Counterstaining• Limitations • current application

Introduction• 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 antigen. • Immunohistochemistry uses antibodies to distinguish

antigenic difference between the cells

Definitions

Immunohistochemistry• 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

Antigens• 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 epitopes.• The outer surfaces of antigens are covered by unique 3-

dimensional protein structures known as epitopes

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. • 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.

Antibody-antigen binding • The associated antibody and antigen are held

together by a combination of hydrogen bonds, electrostatic interactions and van der Waals’ forces.

 

Affinity• 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• 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.

 

Antibody specificity• This refers to the characteristics of an antibody to bind

selectively to a single epitope on an antigen Sensitivity• This refers to the relative amount of antigen that an

immunohistochemical technique is able to detect.

History• 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 and Taylor 1971) has also been

discovered and used to identify immunohistochemical reactions at both light and electron microscopy level.• 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.

Polyclonal 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 dissimilar.PROCEDURE:• 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 immunoglobulin-rich serum. • 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.

Monoclonal antibodies• They are a homogenous population of Ig directed against a single epitope.• Generated by a single B-cell clone from one animal →

immunochemically similar.• Developed by Kohler and Milstein in 1975.PROCEDURE:• 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 specificity.

MONOCLONAL POLYCLONALMouse or rabbit hybridoma Many different species (mostly

rabbits)Tends to be cleaner Tends to have more non-specific

reactivityMore likely to get false- negative results if target epitope is damaged or altered

More likely to have success in an unknown application

Expensive to produce Inexpensive Training is required for the technology used

Skills required are low

Time scale is long for hybridomas Time scale is shortRecognizes only one epitope on an antigen

Recognizes multiple epitopes on ant one antigen

Can produce large amount of specific antibodies

Produces large amounts of non specific antibodies.

Labels

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 microscope• 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 several reasons:

Its small size does not hinder the binding of antibodies to adjacent sites.

Chance of contamination is minimized.Stable enzyme.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 4) Radiolabels

Chromogens • 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

Tissue section

Antigen retrieval

Blocking of endogenous enzyme

Primary antibody

Secondary antibody

Chromogen substrate

Counterstain

Mounting

Microscopic observation

Technique:

Immunohistochemical methodsTraditional 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 cytological preparation.• 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 MethodsPEROXIDASE 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 applications: (1)staining of tissues with high

levels of endogenous peroxidase, (2)double immunostaining in

conjunction with peroxidase, (3)staining of specific cell types

that benefit from the bright red color of alkaline phosphatase substrates

Immunogold silver staining technique (IGSS)• Introduced by Faulk and Taylor (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

Biotin-Avidin Procedure• 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.

Disadvantage: 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.

Advantage: Rapid

(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

Procedure: Application of the primary antibody

subsequent incubations in biotinylated secondary antibody

horseradish peroxidase-labeled streptavidin

subsequent treatment with the biotinylated tyramide amplification reagent.

hydrogen peroxidefree biotin radicals.

These reactive biotin molecules bind covalently to proteins adjacent to the site of the reaction.

Tissue fixation • 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 processing. • 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-fixation. • Ideal fixation is the balance between good morphology and

good antigenicity. • 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 antibody. • 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 constituents.• 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 by formalin.

Unmasking of antigen sitesManual 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 heatingBefore 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 fully exposed. • 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.

Theories:

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 retrieval.• 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

Steamer • 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

Water bath• 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 methods. 

Combined microwave antigen retrieval and trypsin digestion • Infrequently used today; • Brief proteolytic digestion can be carried out before or after

microwave irradiation. 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. Prevention: • 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.Frozen sections • Although the use of frozen sections for diagnostic purposes is

decreasing, immunohistochemistry on frozen sections remains an important histological tool. 

Advantage: preserves enzyme and antigen functionDisadvantages:• 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.

Cytological preparations• 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 false-positive reactions.• Tissues incubated with DAB substrate prior to primary

antibody incubation- if tissue turns brown- peroxidase present and blocking steps needed.• 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 adipocytes.

Prevention:• Addition of a blocking protein, • Addition of a detergent such as Triton 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: • 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 antigen.• 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

COUNTERSTAINING

The final step in the process is counterstaining and mounting slides.counterstains used are:Haematoxylin Hoechst stain and DAPI  (4',6-diamidino-2-phenylindole) Hematoxylin is used as the nuclear counterstain for most routine IHC staining.

• 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 monoclonal antibodies.

3. Antigen loss: The specificity of an antibody for particular antigen and its ability to react with that antigen require the preservation of antigen configuration

Current applications of immunohistochemistry

Tumor Pathology• 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• Amyloidosis• Dementias

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