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- 1. Immunohistochemistry- l Dr SubasMaharjan PG Resident Pathology Department
- 2. INTRODUCTION Definition: • Immunohistochemistry (IHC)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 labelling of antibody or by use of a secondary labelling method.
- 3. Antigens: • it isa foreign substance (protein, carbohydrate or lipid molecule) that bears one or more antibody binding sites. - these sites are composed of a small number of amino acids or monosaccharide units & are known as antigenic determinant groups or epitopes.
- 4. Antibodies: • Also knownas immunoglobulins • Five types: (IgA, IgD, IgE, IgG & IgM) • Formed by plasma cells • Comprises of two light chains and two heavy chains linked by disulphide bonds to form a Y shaped structure.
- 7. History • The principlehas existed since the 1930s. • Started in 1941 when Coons identified pneumococci using a direct fluorescent method. • Indirect method • Addition of horseradish peroxidase • Peroxidase anti-peroxidase technique in 1979 • Use of Avidin & Biotin complex in early 1980’s
- 8. IMMUNOHSITOCHEMISTRY STEPS Slide 3of 23 Tissue sections Antigen retrieval Blocking endogenous enzymes Secondary antibody Primary antibody Microscopy Observation Blocking background staining Chromogen Substrate Counter stain Mounting
- 9. Fixation • To ensurethe preservation of tissue architecture and cell morphology • There is no one universal fixative that is ideal for the demonstration of all antigens
- 10. Coagulant fixative • Organic& nonorganic compound that coagulate protein – render them insoluble • Two types – • Dehydrant (alcohol , acetone) • Strong acid (picric acid, trichloracetic acid) • Histomorphology well maintain but loss of mitochondria and secretary granules
- 11. Cross-Linking Fixatives • Intramolecularand intermolecular cross-links in proteins , formaldehyde (methylol), • Intermolecular bonds in some lipids • Aldehydes – Formaldehyde, Gluteraldehyde,
- 15. Antigen Retrieval • Fixationmodifies the tertiary structure of proteins(Ags) - many times making them undetected by specific Abs • Particularly Necessary when tissues are fixed in cross-linking fixatives. • ‘opening the curtains’ making the section like ‘fresh tissue’ again • Approximately 85% of Ags fixed in formalin require some type of AR to optimize the immunoreaction • Reverse the effect of formalin fixation and re-establish immunoreactivity
- 16. Methods of AntigenRetrieval Heat Induced Epitope Retrieval (HIER)- • Microwave Oven, Pressure Cooker, cooplin jar, water bath, autoclave and Steamer are the most commonly used heating devices. Other devices also include the use of autoclave and water bath • The heating length of 20 minutes appears to be the most satisfactory and the cooling usually takes about 20 minutes
- 17. Microwave antigen retrieval •demonstrated the Ki67 antigen in formalin fixation and paraffin processing. • The method improved the demonstration of well- established antibodies such as CD45 and CD20 • Uneven heating and the production of hot spots have been reported
- 18. Pressure cooker antigenretrieval • more uniform than other heating methods. • unmasking certain nuclear tissue antigens such as bcl-6, p53, p21, estrogen receptor, and progesterone receptor.
- 19. Steamer • Times inexcess of 40 minutes are sometimes required • advantage : less damaging to tissues than the other heating methods. • Commercially available rice steamers are adequate
- 20. Water bath • temperatureto 95–98°C, • advantage of being gentler on the tissue sections because the temperature is set below boiling point. • By using a lower temperature than other heating methods the antigen retrieval buffer does not evaporate • expensive commercial antigen retrieval solutions can be safely reused. • disadvantage in that the antigen retrieval times are increased compared to other methods.
- 21. Autoclave • method offersan alternative form of heat mediated antigen retrieval • good results for nuclear antigens such as MIB1, p21, and p53.
- 22. AR solution • Citratebuffer of pH6.0 is the most popularly used retrieval solution • The TRIS (hydroxymethyl)aminomethane EDTA of pH 10 and EDTA of pH8.0 are second most used retrieval solutions
- 23. Proteolytic InducedEpitope Retrieval (PIER)- • proteinase k, trypsin, chymotrypsin, pepsin, pronase and various other proteases • breaks down formalin cross-linking and the antigenic sites for a number of antibodies are uncovered Combination of Heat Mediated and Proteolytic Enzyme Method- • Alternative approach to unmask antigens if other methods did not work • Especially useful when performing double or triple labeling of two or more antigens simultaneously
- 24. Washing • After almostevery step, except serum blocking, a wash should be performed. This will remove unwanted proteins or chemicals • Wash in Tris(Tris, or trisaminomethane )/ PBS (Phosphate buffered saline)
- 25. Serum blocking • Serumblocking is an important step. The presence of serum proteins keeps the antibodies from binding non-specifically to the slide.
- 26. Hydrophobic and ionicinteraction Collagen, epithelium and adipocytes give rise to background staining due to hydrophobic interactions minimised by addition of a blocking protein or detergent or high salt concentration to buffer • non-immunologically to highly charged groups present on connective tissue elements – innocuous protein solution – Traditionally, non immune serum Major causes of background staining
- 27. – Endogenous enzymeactivity • Fab fragment • ALP • Peroxidase • Biotin
- 28. Biotin Blocking • Ifendogenous biotin is suspected, and especially if using a biotin detection system, use the following method: – Block the slides for 15 minutes in Avidin buffer. • Avidin Buffer: 0.001% Avidin in PBS, pH 7 Use AFTER Primary Antibody Incubation
- 29. Peroxidase Blocking • Ifendogenous levels of peroxidase are suspected, and especially if using a peroxidase detection system: – After primary antibody incubation, block the slides for 10 minutes in peroxidase buffer: • Peroxidase Buffer: 3%H2O2 in PBS
- 30. Detection system • Antibodymolecules must be flagged by some method that permits their visualization. Enzyme labels Colloidal metal labels Fluorescent labels Radiolabels
- 31. • The DirectMethod uses a conjugated primary antibody, and avoids further secondary antibody applications. Direct Method
- 32. IHC – Directmethod • The primary antibody solution should contain antibody, at a concentration recommended by the manufacturer, and diluted in the same buffer as the antibody is already in • Advantages – Rapid and easy to perform • Disadvantages – Helpful only with monoclonal antibody – For each different antigen it is necessary to conjugate each primary antibody separately – High concentration of antibody required – signal strength will always be lower
- 33. The Indirect Methoduses a primary antibody, and then a secondary antibody which is conjugated and recognizes the primary antibody. (The signal may be stronger, because multiple secondaries can recognize one primary.)
- 34. IHC Indirect Method •The secondary antibody should be ‘against’ the animal in which primary antibody was raised in. Should be diluted in the same buffer as primary was diluted in. • After secondary antibody incubation, follow with another wash sequence. • Counter stain, if desired, and wash again. • Apply your detection system, which will react with the conjugation of secondary antibody • Wash again, then dehydrate in 95% EtOH for 1 minute, then 100% EtOH 2X3 minutes. • Clear in Xylene for 2X5 minutes. • Visualize slides as appropriate
- 35. Advantages over directmethod • Increased versatility -Conjugation process applied only to secondary antibody • High specificity • Increased signal strength
- 36. Unlabelled Ab methods •Enzyme bridge technique • Peroxidase-antiperoxidase method • Biotin-avidin procedure • Avidin-biotin conjugate procedure • Biotin streptavidin systems • Polyvalent systems • Alkaline phosphatase anti-alkaline phosphatase method • Protein A methods • Enzyme labelled ag methods • Polymeric labelling two step method • Tyramine signal amplification
- 37. Enzyme bridge technique Asecond Ab is used to link (bridge) the primary Ab to an antiperoxidase Ab, which in turns binds to free peroxidase Avoid the problems inherent in chemical conjugation
- 38. • The PAP(Peroxidase, anti-Peroxidase) Method, uses a third antibody immunologically bound to peroxidase, which recognizes the unconjugated secondary antibody, improving specificity, and signal. • alkaline phosphatase-anti- alkaline phosphatase(APAAP) IHC PAP Method
- 39. • The ABC(Avidin-Biotin Complex) Method exploits the high-affinity binding between avidin and biotin IHC ABC Method
- 40. a crisp, insoluble, stable,dark brown reaction end product
- 41. IHC ABC Method •Different batches of avidin and biotin have differing affinities to each other – drastically affects the sensitivity and reproducibility • Endogenous biotin – nonspecific / false positive staining
- 42. Biotin – StreptavidinSystem • Substitution of streptavidin in the place of avidin • Streptavidin – Tetrameric 60kD avidin analog – Isolated from Streptomyces avidinii – Bind to biotin with a very high affinity
- 43. SP Method (streptavidin peroxidaseconjugated method)
- 44. Advantages over Absystem • Streptavidin contains no carbohydrate which can bind nonspecifically to lectin like substrate • Isoelectric point is closed to neutrality • Highly stable reagent • High sensitivity with modification of the reagents
- 45. Enzyme labels –Horse Radish peroxidase • Why HRP? – Small size – does not hinder binding of antibody to adjacent sites – Easily obtainable in purified form – Stable – Endogenous activity is easily quenched • Chromogen – MC – 3,3’-diaminobenzidene tetrahydrochloride – dark brown reaction product – Although initially reported to be a potential carcinogen, the risk is now thought to be low
- 46. a crisp, insoluble, stable,dark brown reaction end product
- 47. Enzyme Labels –Alkaline phosphatase • Most widely used alternative to HRP – APAAP method using alkaline phosphatase (intestinal) • Endogenous activity? – Blocked by • Levamisole: Inhibits certain types of alkaline phosphatase (not intestinal and placental) • 20% glacial acetic acid: Blocks all types but acid can damage antigens • Chromogen: – Fast red TR (napthol AS-MX phosphate sodium salt) – red
- 48. Enzyme labels -Others • Glucose oxidase – Navy blue reaction – No need for endogenous blocking as animals lack glucose oxidase enzyme • Bacterial B-D-galactosidase – Permanent blue reaction – No need for endogenous blocking as mammalian enzyme requires a different optimal pH for function
- 49. Polymeric labeling twostep method EnVision Systems- • Based on dextran polymer technology • This unique chemistry permits binding of a large number of enzyme molecules (horseradish peroxidase or alkaline phosphatase) to a secondary antibody via the dextran backbone • The benefits are – increased sensitivity, – minimized non-specific background staining – reduction in the total number of assay steps as compared to conventional techniques
- 50. Polymeric labeling twostep method
- 51. CHROMOGENS HORSERADISH PEROXIDASE COLOR SOLUBILITY INALCOHOL DIAMINOBENZIDINE(DAB) BROWN INSOLUBLE DAB WITH ENHANCEMENT BLACK INSOLUBLE 3-AMINO-9-ETHYL CARBAZOLE(AEC) RED TO GREEN BROWN SOLUBLE 4-CHLORO-1-NAPHTHOL(4- CN) BLUE-BLACK SOLUBLE HANKER YATES REAGENT BLUE INSOLUBLE ALPHA NAPHTHOL PYRONIN RED SOLUBLE 3,3’, 5,5’- TETRAMETHYLBENZIDINE(T NBT) BLUE INSOLUBLE
- 52. Counter Staining • Crystalviolet (Methyl violet) stains cell walls purple. • Coomassie Blue stains all protein blue. • Ethidium Bromide binds DNA, and fluoresces orange. • Haematoxylin stains nuclei dark purple. – These are just a few of the stains available!
- 53. ARTIFACTS IN IHC •Many different types of artifacts can be observed in IHC. Some of the more common ones • that I see are listed below. • 1. "Desquamartifact" • 2. "Bubble" artifacts. • 3. Drying artifacts. • 4. Trapping artifacts • 5. Edge artifacts. • 6. Artifacts of inappropriate antibody titers. • 7. Artifacts of poor fixation. • 8. Bacterial contamination artifacts. • 9. Graphite pencil artifacts. • 10. Endogenous biotin artifacts. • 11. Precipitated DAB artifact. • 12. Mold artifact
- 54. Interpretation of Immunohistology Signalpattern- Subcellular localization Four broad categories 1. Membranous pattern 2. Nuclear pattern 3. Cytoplasmic pattern 4. Nuclear-cytoplasmic
- 55.
Editor's Notes
- #4 Hapten From Wikipedia, the free encyclopedia Jump to navigationJump to searchHaptens are minute molecules that elicit an immune response only when attached to a large carrier such as a protein; the carrier may be one that also does not elicit an immune response by itself. (In general, only large molecules, infectious agents, or insoluble foreign matter can elicit an immune response in the body.) Once the body has generated antibodiesto a hapten-carrier adduct, the small-molecule hapten may also be able to bind to the antibody, but it will usually not initiate an immune response; usually only the hapten-carrier adduct can do this. Sometimes the small-molecule hapten can even block immune response to the hapten-carrier adduct by preventing the adduct from binding to the antibody, a process called hapten inhibition. The mechanisms of absence of immune response may vary and involve complex immunological mechanisms, but can include absent or insufficient co-stimulatory signals from antigen-presenting cells. Haptens have been used to study allergic contact dermatitis (ACD) and the mechanisms of inflammatory bowel disease (IBD) to induce autoimmune-like responses.[1] The concept of haptens emerged from the work of Karl Landsteiner[2][3] who also pioneered the use of synthetic haptens to study immunochemical phenomena.[4] Examples of haptens[edit] The first researched haptens were aniline and its carboxyl derivatives (o-, m-, and p-aminobenzoic acid).[5] A well-known example of a hapten is urushiol, which is the toxin found in poison ivy. When absorbed through the skin from a poison ivy plant, urushiol undergoes oxidation in the skin cells to generate the actual hapten, a reactive quinone-type molecule, which then reacts with skin proteins to form hapten adducts. Usually, the first exposure causes only sensitization, in which there is a proliferation of effector T-cells. After a subsequent, second exposure, the proliferated T-cells can become activated, generating an immune reaction that produces typical blisters of a poison ivy exposure. Some haptens can induce autoimmune disease. An example is hydralazine, a blood pressure-lowering drug that occasionally can produce drug-induced lupus erythematosus in certain individuals. This also appears to be the mechanism by which the anaesthetic gas halothane can cause a life-threatening hepatitis, as well as the mechanism by which penicillin-class drugs cause autoimmune hemolytic anemia. Other haptens that are commonly used in molecular biology applications include fluorescein, biotin, digoxigenin, and dinitrophenol. Lastly, nickel allergy is caused by nickel metal ions penetrating the skin and binding to skin proteins. Hapten Conjugation
- #5 Epitope From Wikipedia, the free encyclopedia Jump to navigationJump to searchAn epitope, also known as antigenic determinant, is the part of an antigen that is recognized by the immune system, specifically by antibodies, B cells, or T cells. For example, the epitope is the specific piece of the antigen to which an antibody binds. The part of an antibody that binds to the epitope is called a paratope. Although epitopes are usually non-self proteins, sequences derived from the host that can be recognized (as in the case of autoimmune diseases) are also epitopes. The epitopes of protein antigens are divided into two categories, conformational epitopes and linear epitopes, based on their structure and interaction with the paratope.[1] A conformational epitope is composed of discontinuous sections of the antigen's amino acid sequence. These epitopes interact with the paratope based on the 3-D surface features and shape or tertiary structure of the antigen. The proportion of epitopes that are conformational is unknown.[citation needed] By contrast, linear epitopes interact with the paratope based on their primary structure. A linear epitope is formed by a continuous sequence of amino acids from the antigen. T cell epitopes[edit] T cell epitopes are presented on the surface of an antigen-presenting cell, where they are bound to MHC molecules. In humans, professional antigen-presenting cells are specialized to present MHC class II peptides, whereas most nucleated somatic cells present MHC class I peptides. T cell epitopes presented by MHC class I molecules are typically peptides between 8 and 11 amino acids in length, whereas MHC class II molecules present longer peptides, 13-17 amino acids in length,[2] and non-classical MHC molecules also present non-peptidic epitopes such as glycolipids. Cross-activity[edit] Epitopes are sometimes cross-reactive. This property is exploited by the immune system in regulation by anti-idiotypic antibodies (originally proposed by Nobel laureate Niels Kaj Jerne). If an antibody binds to an antigen's epitope, the paratope could become the epitope for another antibody that will then bind to it. If this second antibody is of IgM class, its binding can upregulate the immune response; if the second antibody is of IgG class, its binding can downregulate the immune response.[citation needed] B cell Epitopes
- #10 cross-linking mechanism involving the amino groups of lysine and other amino acids through the formation of pyridine intermediaries. The use of osmium tetroxide fixation to preserve lipids is fundamental to TEM
- #18 volume of buffer between 400 and 600 ml in a suitably sized microwave-resistant plastic container,
- #19 A pressure cooker at 15 psi (10.3 kPa) reaches a temperature of around 120°C at full pressure.
- #23 ethylenediaminetetraacetic acid (EDTA)
- #28 3,3α-diaminobenzidine tetrahydrochloride (DAB),
- #29 Pyruvate carboxylase, an enzyme in the Krebs cycle, contains four molecules of biotin, and is localized in mitochondria within the cytoplasm of cells. Considering this fact, it is not surprising that endogenous biotin artifact is most prominent in metabolically active cells that contain numerous mitochondria, such as liver and kidney cells. As
- #32 alp is sued in hematological cases n skin as melanocyte also contains browm color fluorescein gree and rhodamine red strepta vidin ha good affinity for biotin but poory express its all 4 sites biotin has isoelectric property so that it can bind to negative charge protein like lectin
- #40 Pyruvate carboxylase, an enzyme in the Krebs cycle, contains four molecules of biotin, and is localized in mitochondria within the cytoplasm of cells. Considering this fact, it is not surprising that endogenous biotin artifact is most prominent in metabolically active cells that contain numerous mitochondria, such as liver and kidney cells. As
- #41 Other chromogens are available, including: 3-amino-9-ethylcarbazole (Graham et al., 1965; Kaplow, 1975), which gives a red final reaction product; 4-chloro-1-naphthol (Nakane, 1968), a blue final reaction product; Hanker-Yates reagent (Hanker et al., 1977), a dark blue product and α-naphthol pyronin (Taylor & Burns, 1974), a red-purple final reaction product. Many of these contain hazardous reagents and have now largely been superseded by commercial chromogens available in kit form. Vector Laboratories, for example produce a wide range of different colored chromogens suitable as alternatives to DAB which can be used for multilabeling techniques. These include Vector Red, Vector Blue, Vector VIP (purple) and BCIP/NBT(blue/ violet).
- #47 Other chromogens are available, including: 3-amino-9-ethylcarbazole (Graham et al., 1965; Kaplow, 1975), which gives a red final reaction product; 4-chloro-1-naphthol (Nakane, 1968), a blue final reaction product; Hanker-Yates reagent (Hanker et al., 1977), a dark blue product and α-naphthol pyronin (Taylor & Burns, 1974), a red-purple final reaction product. Many of these contain hazardous reagents and have now largely been superseded by commercial chromogens available in kit form. Vector Laboratories, for example produce a wide range of different colored chromogens suitable as alternatives to DAB which can be used for multilabeling techniques. These include Vector Red, Vector Blue, Vector VIP (purple) and BCIP/NBT(blue/ violet).
- #48 soluble in alcohol – requires aqueous mount (eg. neutral phosphate buffered glycerin jelly)
- #49 Glucose oxidase - fungus