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![History
1942
• Coons, Creech, Jones, Berliner: Developed indirect immunofluorescence
method to demonstrate pneumococcal antigen in tissue
1966
• Graham & Karnovsky, Developed enzyme tagging method [horseradish
peroxidase]
1967
• Nakane & Pierce, Developed enzyme-labelled antibody technique
[immunoperoxidase]
1970
• Sternberger, Developed unlabelled antibody method [peroxidase-
antiperoxidase (PAP) technique]
1974
• Heitzman & Richards, Developed another unlabelled antibody method
[avidin-antibiotin complex (ABC) technique]
1996
• Dako developed polymeric techniques like EPOS and more recently
EnVision detection system](https://image.slidesharecdn.com/ihcshn-180308162609/85/Immunohistochemistry-3-320.jpg)
![History
1942
• Coons, Creech, Jones, Berliner: Developed indirect immunofluorescence
method to demonstrate pneumococcal antigen in tissue
1966
• Graham & Karnovsky, Developed enzyme tagging method [horseradish
peroxidase]
1967
• Nakane & Pierce, Developed enzyme-labelled antibody technique
[immunoperoxidase]
1970
• Sternberger, Developed unlabelled antibody method [peroxidase-
antiperoxidase (PAP) technique]
1974
• Heitzman & Richards, Developed another unlabelled antibody method
[avidin-antibiotin complex (ABC) technique]
1996
• Dako developed polymeric techniques like EPOS and more recently
EnVision detection system](https://image.slidesharecdn.com/ihcshn-180308162609/75/Immunohistochemistry-3-2048.jpg)
More Related Content
Similar to Immunohistochemistry
Immunohistochemistry
- 1. IMMUNOHISTOCHEMISTRY Dr. Shankar Bastakoti JR,Pathology 3rd year
- 2. • Technique foridentifying antigens in tissue (‘histo’) by means of antigen-antibody interactions (‘immuno- chemistry’) • The advent of diagnostic immunohistochemistry has made such an impact so known as ‘brown revolution’ and as ‘magic markers’.
- 3. History 1942 • Coons, Creech,Jones, Berliner: Developed indirect immunofluorescence method to demonstrate pneumococcal antigen in tissue 1966 • Graham & Karnovsky, Developed enzyme tagging method [horseradish peroxidase] 1967 • Nakane & Pierce, Developed enzyme-labelled antibody technique [immunoperoxidase] 1970 • Sternberger, Developed unlabelled antibody method [peroxidase- antiperoxidase (PAP) technique] 1974 • Heitzman & Richards, Developed another unlabelled antibody method [avidin-antibiotin complex (ABC) technique] 1996 • Dako developed polymeric techniques like EPOS and more recently EnVision detection system
- 4. Principle • Immunohistochemistry :detecting the presence of specific proteins (antigen) in cells or tissues Existence and distribution of an antigen detected micro chemically is made manifest macro chemically • Sharp localization of target components in cell and tissue is based on a satisfactory signal-to-noise ratio Amplifying the signal while reducing the nonspecific background staining(noise) is the major strategy Immunohistochemistry is the localization of antigens or proteins in tissue sections by the use of labeled antibodies as specific reagents through antigen-antibody interactions that are visualized by a marker such as fluorescent dye, enzymes etc.
- 5. Structure of Protein(antigen) • Antigen is a molecule that induces the formation of an antibody. • Bears one or more antibody binding sites. • Epitopes/ antigenic determinant groups, composed of small number of aminoacids or monosaccharide units form highly specific topographic sites for antibody binding. Antibody structure - monomer IgG is the commonest and most frequently used antibody for IHC
- 6. Antigen – Antibodybinding Binding • AA side chains of variable domain of antibody forms a cavity, geometrically and chemically complementary to a single type of antigen epitope • The associated antibody and antigen are held together by a combination of hydrogen bonds, electrostatic forces and Van der Waals’ forces Factors in an Ag-Ab reaction • Affinity: 3D fit of antibody to the antigen epitope • Measure of the binding strength • Avidity: Multivalency of the antibody • Multiple antibodies reacting with different epitopes of the same antigen are less likely to be removed by washing • Functional combining strength • Specificity • Sensitivity
- 7. Primary antibodies Polyclonal antibodies •Obtained by immunising an animal with a purified specific immunogen (antigen of interest) and harvested by bleeding to obtain immunoglobulin rich serum • Mixture of antibodies to different epitopes . • Also contain antibodies to impurities on the immunogen (usually of low titre and affinity) • May cross react with other molecules • SENSTIVE BUT LESS SPECIFIC – more background staining Monoclonal antibodies • Hybridoma technique by Kohler and Milstein (1975) • Constant reliable production of one pure antibody with one epitope specificity • Cross reactivity to other molecules can be eliminated by screening and careful selection of hybrids • Dramatically increased the number of antibodies available for IHC, particularly in paraffin embedded sections Lectins • Plant or animal proteins that bind tissue carbohydrates with high specificity
- 8. Direct method • Primaryantibody is conjugated directly to the label • Little signal amplification and lower sensitivity • Mainly used in demonstration of Ig and complement in frozen sections of skin and renal biopsies Indirect method • Primary antibody is unlabelled • Secondary antibody directed against the immunoglobulin is conjugated with the label • More signal amplification and higher sensitivity • Versatile • Same secondary antibody can be used for a multiple primary antibodies of the same animal species
- 9. LABELS • Enzyme labels •Enzyme incubated with chromogen produces a stable coloured reaction end product • Enzymes: • MC used - Horse Radish peroxidase with chromogen DAB • Calf intestinal alkaline phosphatase • Glucose oxidase • Bacterial derived B-D-galactosidase • Colloidal metal labels • Colloidal gold alone or with silver precipitation to amplify visibility and enhanced using polarised incidental light. • Fluorescent labels • Radiolabels • Although requires auto radiographic facilities, it can quantify reaction • Limitation of availability of internally labeled antibodies
- 10. 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. • Endogenous activity? • Endogenous peroxidase activity is present in neutrophils and myeloid cells • Blocking procedures, most popular is hydrogen peroxide-methanol method • Chromogen • MC – 3,3’-diaminobenzidene tetrahydrochloride – dark brown reaction product • Initially reported to be a potential carcinogen, the risk is now thought to be low.
- 11. Endogenous peroxidase blocking •Most commonly used method: • Incubation in absolute methanol containing 0.5% hydrogen peroxide for 10 minutes in room temperature • Produce almost complete abolition without affecting immunoreactivity • Can be done before or after primary antibody incubation. Generally blocking after primary antibody is recommended as it may interfere with binding of primary antibody to antigen
- 12. Immunohistochemical methods Direct technique •Traditional direct technique: • Hardly used except in skin and kidney using more commonly fluorochrome conjugates (IF) • Quick and easy • BUT • Helpful only with monoclonal antibody • For each different antigen it is necessary to conjugate each primary antibody separately • High concentration of antibody required • New direct technique: • EPOS – Enhanced Polymer One Step staining • A large number of primary antibody molecules and peroxidase enzymes are attached to a dextan ‘polymer’ blackbone • Signal amplification is increased and more sensitivity • Time duration reduced • However, limited number of such primary antibodies available FITC FITCFITC FITC FITC FITC FITC FITC FITC
- 13. Immunohistochemical methods Indirect technique •Two Step indirect technique • Polymer chain two step indirect technique • Unlabeled antibody-enzyme complex techniques HRP HRP HRP HRP HRP HRP HRP
- 14. Polymer chain twostep technique • Unconjugated primary antibody followed by a secondary antibody conjugated to a labeled polymer dextran chain • 70 molecules of the label (HRP enzyme) and 10 molecules of secondary antibody • Polymer may contain both anti-mouse and anti-rabbit IgG and therefore can be used for both mouse and rabbit primary antibodies • Biotin free • Quick, reliable and reproducible method
- 15. Unlabelled antibody-enzyme complex techniques(PAP and APAAP) • PAP (peroxidase- antiperoxidase) - three step • Unconjugated primary antibody • Unconjugated second layer of bridging antibody which has binding sites for both primary antibody and third layer complex • Third layer of PAP complex – 3 peroxidase molecules and 2 anti-peroxidase molecules • APAAP technique • Alkaline phosphatase antibodies raised in mouse can be used by the same principle • These methods have been largely replaced by ABC and polymer methods
- 16. Immunohistochemical methods (Strept)avidin-biotin technique(ABC) • Most widely used methodology in diagnostic IHC along with indirect polymer techniques • Three step technique • These methods rely on the marked affinity of the basic glycoprotein avidin for the small water soluble vitamin biotin (Vit H) • Disadvantages of avidin: • High isoelectric pH of 10; therefore it has + charge at neutral pH leading to non specific binding with negative charged structures such as nuclei • Being a glycoprotein, it can react with carbohydrate moiety of lectins leading to non specific reactions • Substitution of streptavidin overcomes these problems • Streptavidin isolated from bacterium Streptomyces avidini • 4 high affinity binding sites for Biotin • Streptavidin contains no carbohydrate which can bind nonspecifically to lectin like substrate • Isoelectric point is closed to neutrality • Highly stable reagent • Biotin • Biotin is easily conjugated to antibodies and enzymes • Up to 150 biotin molecules can be attached to one antibody molecule
- 17. (Strept)avidin-biotin technique (ABC) •The ABC technique can employ either enzyme bound directly to the streptavidin • Alternatively, both secondary antibody and the enzyme can be biotin labelled and streptavidin acts as a bridge between them Biotin Biotin Biotin Biotin Biotin Biotin Biotin Biotin BiotinA vidin HRP A vidin HRP A vidin HRP A vidin HRP A vidin HRP A vidin HRP A vidin HRP A vidin HRP
- 18. (Strept)avidin-biotin technique (ABC) Endogenousbiotin blocking • Tissues rich in endogenous biotin such as liver and kidney will require the use of avidin/biotin block before applying primary antibody • Block the slides for 15 minutes in Avidin buffer. • Avidin Buffer: 0.001% Avidin in 0.1M PBS, pH 7 • Wash in buffered Tris saline after blocking
- 19. STEPS OF IHC Slide3 of 23 Tissue sections Antigen retrieval Blocking endogenous enzymes Secondary antibody Primary antibody Microscopy Observation Blocking background staining Chromogen Substrate Counter stain Mounting
- 20. Step 1: Deparaffinization •Generally polylysine (0.1% solution) coated or charged slides are used to ensure that the sections are adherent to the slide • This step removes the paraffin still remaining on slide, which would otherwise interfere with the ability of the antibodies to detect the proteins of interest. Paraffin Proteins
- 21. Step 2: AntigenRetrieval • Fixation modifies the tertiary structure of proteins(Ags) - many times making them undetected by specific Abs • Cross-linking fixatives like formalin lead to formation of methylene bridges which cross-link proteins and therefore mask antigenic sites • Cross-linking the irrelevant protein to the peptide epitopes sterically blocked antibodies from binding • The epitopes for clinical antibodies require only primary protein structure • Antigen retrieval dissociates irrelevant proteins and restores immunoreactivity by probably denaturing the secondary and tertiary structure of the irrelevant protein.
- 22. Methods of AntigenRetrieval •Microwave Oven, Pressure Cooker 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 Heat Induced Epitope Retrieval (HIER)- •Proteinase k, trypsin, chymotrypsin, pepsin, pronase and various other proteases Proteolytic Induced Epitope Retrieval (PIER)- •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 Combination of Heat Mediated and Proteolytic Enzyme Method-
- 23. Heat induced epitoperetrieval Two factors are important: • PH value of AR solution • Most antigens – no significant variation in pH value ranging from 1.0-10.0 • 0.01 M Citrate buffer of pH 6.0 is the most popularly used retrieval solution • The TRIS-EDTA of pH 9.0 and 0.1mM EDTA of pH 8.0 are second most used retrieval solutions • 0.01 M TRIS-HCl, pH 1 or 10, was slightly superior to citrate buffer of pH 6.0 and gave the best overall results . • Heating temp and heating time- • The relationship between the temperature and time is inverse • Temperature below 80*c – not significantly beneficial
- 24. A New Methodfor Unmasking Antigens • LAB Solution (liberating antibody binding) • Thiocyanic acid, compd. with guanidine 1 (1:1) + Water • An effective method for routine unmasking of antigens for IHC staining based on simple protein chemistry • Allows antigen unmasking in less time and at room temperature or much lower temperatures than other methods • Can be used on automated stainers whether they have heating capabilities or not .
- 25. Step 3: Blocking •Major causes of background staining • Hydrophobic and ionic interactions • Hydrophobic interactions are the result of cross linking of amino acids both within and between adjacent protein molecules due to aldehyde fixation • Collagen, epithelium and adipocytes give rise to background staining due to hydrophobic interactions • Non specific staining is most commonly produced because the primary antibody is attracted non-immunologically to highly charged groups present on connective tissue elements. • Endogenous enzyme activity • Peroxidase blocking • Biotin blocking
- 26. Serum Blocking • Serumblocking is an important step. The presence of serum proteins keeps the antibodies from binding non-specifically to the slide and reduces non specific background staining. Do not wash after serum blocking. • Endogenous enzyme blocking for peroxidase x10 minutes • Biotin block, if necessary Serum Proteins
- 27. Step 4: Washing •After almost every step, except serum blocking, a wash should be performed. This will remove unwanted proteins or chemicals • Wash in Tris buffered saline Unbound Proteins WASH
- 28. Step 5: Primaryantibody incubation • For optimum staining, primary antibody is to be used at correct dilution. Too high and too low concentrations may cause false negative results • Generally, around 100 µl is the quantity of diluted primary antibody required for a one section • A moist chamber • Time for incubation depends on the individual antibody varying from one hour to overnight incubation • Wash in TRIS buffered saline
- 29. Step 6: Secondaryantibody incubation • In a standard 3 step ABC protocol • Incubate with secondary reagent (biotinylated goat anti-rabbit immunoglobulins) followed by buffer rinse • Incubate slides with ABC complex conjugated with HRP followed by buffer rinse • In a two step HRP polymer method • HRP polymer secondary antibody incubated in a single step
- 30. Step 7: Detectionof immune reaction by chromogen • Addition of DAB reagent or other chromogen for 5- 15 minutes • Stop reaction by bringing sections to deionized water
- 31. Step 8: CounterStaining • Counterstain with Mayer’s hematoxylin (dark purple) • Dehydrate and coverslip
- 32. What we dohere!!! What Aiimsians do!!! Tissue sections 3 to 4 micro thickness on APES (3-aminopropyl triethoxy silane) coated slides. Tissue sections 3 to 4 micro thickness on APES coated slides. Slides are left in hot plate for few minutes and later dipped in xylene (2 change, 5 mins each). Slides are left in hot plate for few minutes and later dipped in xylene (2 change, 5 mins each). Slides are then rehydrated through acetone (two change, 5 mins each), alcohol for 5 mins and running tap water for 5 mins. Slides are then rehydrated through acetone (1 change, 5 mins ), alcohol for 5 mins and running tap water and distilled water for 5 mins. Slides are not allowed to dry at any point. Slides are not allowed to dry at any point. Antigen retrieval is performed by placing slides in citrate buffer in pressure cooker for 15 mins. Antigen retrieval by placing slides in citrate buffer in microwave oven at 100⁰ C for 30mins (100 C = 900 W in microwave)
- 33. Slides are cooledat room temperature and next steps of staining are performed in moist chamber Slides are cooled at room temperature and next steps of staining were performed in moist chamber. Slides are washed with wash buffer (Tris buffer solution). The slides are washed with running tap water. Slides are incubated in Hydrogen peroxide block (3% hydrogen peroxide in methanol) for 10 mins. Slides are incubated in Hydrogen peroxide block (4% hydrogen peroxide) for30 mins. Slides are washed with Tris buffer (4 changes, 5 mins each). Slides are washed with running tap water and distilled water and 1 change of Tris buffer. The primary antibody is added and kept overnight in freeze. Ultravision protein block for 5 mins at room temperature (100 ml of Tris buffer with 0.5 ml of protein block)
- 34. Washed with washbuffer and super enhancer (secondary antibody, Ultravision One HRP Polymer) is applied for 30 mins and washed with wash buffer 3 times. Washed with Tris buffer (4 changes, 5 mins each).Marked with pap pen (paraffin pen) The primary are added and incubated at room temperature for 2 hrs. DAB added to slide for 10-15 mins and washed with Tris buffer. Slides were washed with wash buffer ( 4 changes). Primary antibody amplifier quanto is added and kept for 15 mins The slides are dipped in Harris hematoxylin for 30 secs and washed in running tap water for 5 mins. HRP polymer quanto is added and kept for 30 mins. The slides are washed with tris buffer (4 changes). DAB added to slide for 3 mins and washed with running water. Dipped in hematoxylin for 30 secs and washed in running tap water for 5 mins. The slides are air dried and mounted with diphenylene phthalate xylene The slides are air dried and mounted with DPX.
- 37. Trouble shooting • Falsenegative • Incomplete deparaffinization • Incorrect retrieval solution • Inadequate heat induced epitope retrieval temperature • Extreme digestion using enzyme retreival • Wrong antibody concentration • Incorrect chromogen • Wrong positive control • Human or machine error
- 38. Trouble shooting • Falsepositive staining • Poor fixation • Poor technical quality of sections • Variation in pH of retrieval solution • Endogenous enzyme activity and inadequate blocking • Species cross reactivity • Tissue drying (edge artifacts) • Chromogen precipitation and streaking • Automation error usually incomplete rinsing
- 39. ABSENT STAINING ORWEAK STAINING PROBLEM SOLUTION INADEQUATE FIXATION Avoid delay of fixation( >30 min) or overfixation (>48 hrs) INCOMPLETE DEHYDRATION DURING PROCESSING Perform regular reagent changes(i.e. Alcohol) PARAFFIN TOO HOT (heat sensitive epitopes lost) Monitor temp of paraffin(<56⁰C) PROLONGED OR EXCESSIVE HEATING Optimize AR time ANTIBODY TOO DILUTE Check Ab titration; increase conc ; lenghten incubation time; increase temp of Rxn DRYING OUT OF TISSUE DURING PROCESSING Keep specimen moist; use humidity chamber INSUFFICIENT INCUBATION TIME Lengthen incubation time; add heat; increase conc of Ab CHROMOGEN NOT WORKING Add chromogen to labelling solution and monitor for change in color REAGENT NOT WORKING Check expiry, pH and compatibility with other reagents
- 40. BACKGROUND STAINING PROBLEM SOLUTION NONSPECIFICPROTEIN BINDING Use nonimmune serum from same animal species as sec Ab; add salt to buffer POORLY FIXED OR NECROTIC TISSUE Make sure tissue is properly fixed; avoid sampling of necrotic areas THICK PREPARATION Cut sections at 3 to 5 mm INAPPROPRIATELY CONC Ab Check titration; decrease conc; decrease incubation time; decrease temp of Rxn ENDOGENOUS BIOTIN Block with avidin INCOMPLETE RINSING OF SLIDES Follow the protocol for proper slide rinsing CHROMOGEN STAINING TOO INTENSE Monitor timing of chromogen substrate Rxn; filter the chromogen; decrease the chromogen conc.
- 41. Common applications of Immunohistochemistry •Characterization of Poorly differentiated malignancy • Differentiating in-situ lesion from invasive malignancy • Metastatic tumour work up • Determination of viral etiology • Prognostic and therapeutic value
- 42. Interpretation of Immunohistology Signalpattern: Sub-cellular localization Four broad categories 1. Membranous pattern 2. Nuclear pattern 3. Cytoplasmic pattern 4. Nuclear-cytoplasmic
- 43.
Editor's Notes
- #12 APAAP method using alkaline phosphatase (intestinal, Glucose oxidase, Bacterial B-D-galactosidase
- #24 Other exhibit better results at very acidic or basic pH Nuclear antigens(MIB1, ER)- Decrease intensity in middle range pH value with optimal results at low pH HMB45 – negative / weak staining in low pH and excellent results in high pH range
- #26 The most effective way is to add an innocuous protein solution to the section before primary antibody which will saturate and neutralise the charged sites Traditionally, non immune serum from the animal species in which the second bridging antibody is raised is used as a blocking serum.