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PRESENTED BY:
DR KALPAJYOTI BHATTACHARJEE
IMMUNOHISTOCHEMISTRY METHODS
Contents
• Introduction
• Definitions
• History
• Principle
• Production of primary reagents
• Technique
• Immonohistochem...
Introduction
• Immunohistochemistry is as the name implies, a combination of two
disciplines – immunology and histology. T...
Definitions
Immunohistochemistry
• This is a technique for identifying cellular or tissue constituents
(antigens) by means...
Antigens
• An antigen is a molecule that induces the formation of an antibody and
bears one or more antibody binding sites...
Antibody
• Antibodies belong to the class of serum proteins known as immunoglobulins.
The terms antibody and immunoglobuli...
Antibody-antigen binding
• The associated antibody and antigen are held together by a
combination of hydrogen bonds, elect...
Affinity
• Affinity is the three-dimensional fit of the antibody to its specific antigen
and is a measure of the binding s...
Antibody specificity
• This refers to the characteristics of an antibody to bind selectively to a
single epitope on an ant...
History
• The principle has existed since the 1930s.
• Started in 1941 when Albert H. Coons et al tagged fluorescein dye t...
Principle of Immunohistochemistry:
• Immunohistochemistry is a method for localizing specific antigen in
tissues or cells ...
Production of primary reagents
• It is the pivotal reagent common all IHC techniques.
• They impart specificty.
• Two type...
Polyclonal antibodies
• They are a heterogenous mixture of antibodies directed against various epitopes
of same antigen.
•...
Monoclonal antibodies
• They are a homogenous population of Ig directed against a single epitope.
• Generated by a single ...
MONOCLONAL POLYCLONAL
Mouse or rabbit hybridoma Many different species (mostly rabbits)
Tends to be cleaner Tends to have ...
Labels
1) Enzyme labels
• Enzymes are the most widely used labels in immunohistochemistry, and
incubation with a chromogen...
• Horseradish peroxidase is commonly used as an antibody label for
several reasons:
Its small size does not hinder the bi...
• Calf intestinal alkaline phosphatase is the most widely used
alternative enzyme tracer to horseradish peroxidase, partic...
2) Colloidal metal labels
• When used alone, colloidal gold conjugates appear pink when viewed
using the light microscope....
Chromogens
• 3,3α-diaminobenzidene tetrahydrochloride (DAB), it yields a crisp,
insoluble, stable, dark brown reaction end...
Tissue section
Antigen retrieval
Blocking of endogenous enzyme
Primary antibody
Secondary antibody
Chromogen substrate
Cou...
Immunohistochemical methods
Traditional direct technique
• The primary antibody is conjugated directly to the label. The c...
New Direct technique
• Pluzek et al in 1993
• Commercial name- Enhanced Polymer One-step Staining (EPOS)
• A large number ...
Two-step indirect technique
• A labeled secondary antibody directed against the immunoglobulin of
the animal species in wh...
Polymer chain two-step indirect technique
• This technology uses an unconjugated primary antibody, followed by a
secondary...
Unlabeled Antibody Methods
PEROXIDASE ANTIPEROXIDASE METHOD / PAP
• Immune complex typically consists of 2 antibody molecu...
ALKALINE PHOSPHATASE–ANTIALKALINE PHOSPHATASE
METHOD / APAAP
• Principle same as those described for the PAP method except...
Immunogold silver staining technique (IGSS)
• Introduced by Faulk andTaylor (1971).
• In this method the gold particles ar...
Biotin-Avidin Procedure
• The biotin-avidin procedure exploits the high affinity binding between
biotin and avidin.
Biotin...
Disadvantage:
1) Different batches of biotin and different batches of avidin have
differing affinities for one other → aff...
(Strept) avidin-biotin techniques
• The labeled streptavidin-biotin technique is the most widely used
methodology in diagn...
• 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
sig...
Procedure:
Application of the primary antibody
subsequent incubations in biotinylated secondary
antibody
horseradish perox...
Tissue fixation
• A prerequisite for all routine histological and cytological investigations is
to ensure preservation of ...
• Show good preservation of morphologic details after embedding in a
support medium (e.g., paraffin).
• Good fixation is t...
• Fixative can cause changes in the steric configuration of proteins, which
may mask antigenic sites (epitopes) and advers...
• The most popular choice of fixatives for routine histology are formalin
based, either as a 10% solution or with the addi...
Advantages of formalin:
• good preservation of morphology, even after prolonged fixation
• economical chemical
• sterilize...
Unmasking of antigen sites
Manual methods for antigen unmasking include:
• Proteolytic enzyme digestion
• Microwave oven i...
Proteolytic enzyme digestion
• Described by Huang et al. (1976), Curran and Gregory (1977), and
Mepham et al. (1979).
• Th...
Heat-mediated antigen retrieval techniques
• Heat-based antigen retrieval methods have brought a great improvement in
the ...
During formalin fixation intermolecular and methylene bridges and weak
Schiff bases form intramolecular cross-linkages, wh...
Microwave antigen retrieval
• Shi et al. (1991) first established the use of microwave heating for antigen
retrieval.
• Ge...
Pressure cooker antigen retrieval
• Norton et al. (1994) suggested the use of the pressure cooker as an
alternative to the...
Steamer
• Steam heating appears to be less efficient than either microwave oven
heating or pressure cooking
• Advantage - ...
Water bath
• Kawai et al. (1994) demonstrated that a water bath set at 90°C was
adequate for antigen retrieval.
• Increasi...
Combined microwave antigen retrieval and trypsin digestion
• Infrequently used today;
• Brief proteolytic digestion can be...
Pitfalls of heat pretreatment
• Care should be taken not to allow the sections to dry after heating, as
this destroys anti...
Commercial antigen retrieval solutions
• There are numerous commercial antigen retrieval solutions available.They
can be e...
Immunohistochemistry in practice
• The choice of technique to suit the needs of particular types of work is
governed by so...
• Poor morphology → improved by ensuring the frozen sections are thoroughly
dried both before and after the sections are f...
Cytological preparations
• Acetone-fixed smears are often preferred by the immunohistochemist as
it allows a wide range of...
Blocking endogenous enzymes
• If enzymes similar to those used as the antibody label are present in the
tissue, they may r...
• Most endogenous alkaline phosphatase activity can be blocked by:
Adding levamisole in the final incubating medium.
Usi...
Blocking background staining
• The major causes of background staining in immunohistochemistry are
hydrophobic and ionic ...
Hydrophobic interactions :
• Tissues that give background staining as a result of hydrophobic
interactions include collage...
• Non-specific staining is most commonly produced because the primary
antibody is attracted nonimmunologically to highly c...
Controls:
• Controls validate immunohistochemical results.
• It is essential that any method using immunohistochemistry pr...
COUNTERSTAINING
The final step in the process is counterstaining and mounting slides.
counterstains used are:Haematoxylin
...
• Hoechst stains are part of a family of blue fluorescent dyes used
to stain DNA. There are three related Hoechst stains: ...
Limitations of Immunohistochemistry:
1. Experience: Experience is critical in standardizing the procedure
including the se...
Current applications of immunohistochemistry
Tumor Pathology
• Classification of Neoplasma
• Diagnosis of Malignancy
• Pro...
Thank you
Immunohistochemistry  METHODS
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Immunohistochemistry METHODS

ITS ALL ABOUT BASICS OF IHC. ANTIGEN ANTIBODIES TECHNIQUES PROCEDURES ETC....

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Immunohistochemistry METHODS

  1. 1. PRESENTED BY: DR KALPAJYOTI BHATTACHARJEE IMMUNOHISTOCHEMISTRY METHODS
  2. 2. 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
  3. 3. 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
  4. 4. 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
  5. 5. 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
  6. 6. 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.
  7. 7. Antibody-antigen binding • The associated antibody and antigen are held together by a combination of hydrogen bonds, electrostatic interactions and van der Waals’ forces.
  8. 8. 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.
  9. 9. 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.
  10. 10. 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 andTaylor 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.
  11. 11. 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.
  12. 12. 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.
  13. 13. 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.
  14. 14. 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.
  15. 15. MONOCLONAL POLYCLONAL 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 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 short Recognizes 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.
  16. 16. 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).
  17. 17. • 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.
  18. 18. • 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
  19. 19. 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
  20. 20. 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
  21. 21. Tissue section Antigen retrieval Blocking of endogenous enzyme Primary antibody Secondary antibody Chromogen substrate Counterstain Mounting Microscopic observation Technique:
  22. 22. Immunohistochemical methods 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 cytological preparation. • Quick and easy to use. • Provides little signal amplification • Lacks the sensitivity.
  23. 23. 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.
  24. 24. 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.
  25. 25. 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.
  26. 26. 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.
  27. 27. 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
  28. 28. 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
  29. 29. 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.
  30. 30. 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
  31. 31. (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
  32. 32. • Streptavidin has now largely replaced the use of avidin in immunohistochemical detection techniques.
  33. 33. 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
  34. 34. 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 peroxide free biotin radicals. These reactive biotin molecules bind covalently to proteins adjacent to the site of the reaction.
  35. 35. 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.
  36. 36. • 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.
  37. 37. • 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.
  38. 38. • 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.
  39. 39. 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.
  40. 40. 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.
  41. 41. 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.
  42. 42. 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
  43. 43. 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
  44. 44. 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
  45. 45. 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
  46. 46. 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
  47. 47. 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.
  48. 48. 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.
  49. 49. 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.
  50. 50. 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
  51. 51. 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 function Disadvantages: • Poor morphology • Limited prospective studies • Storage of material difficult • Cutting difficulty over paraffin sections
  52. 52. • 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.
  53. 53. 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.
  54. 54. 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.
  55. 55. • 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
  56. 56. 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.
  57. 57. 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 asTriton X , • Addition of a high salt concentration, 2.5% NaCl, to the buffer. • Addiion of the blocking serum to the diluted primary antibody.
  58. 58. • 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.
  59. 59. 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
  60. 60. 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.
  61. 61. • 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,
  62. 62. 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
  63. 63. 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
  64. 64. Thank you

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