Immunohistochemistry

Histopathology

1. Immunohistochemistry

2. Immunohistochemistry 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. Definition ; 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.Im munohistochemistry- using tissue sections. Immunocytochemistry - cytological preparations

3. Continuous.. Antigen ; In immunology, an antigen is a molecule or molecular structure or any foreign particulate matter or a pollen grain that can bind to a specific antibody or T-cell receptor. The presence of antigens in the body may trigger an immune response. Antibody ; An antibody, also known as an immunoglobulin, is a large, Y-shaped protein used by the immune system to identify and neutralize foreign objects such as pathogenic bacteria and viruses. The antibody recognizes a unique molecule of the pathogen, called an antigen.

4. Continuous 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.

5. Principle of immunohistochemistry 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.

6. IHC sample preparation Sample preparation: IHC can be broadly classified into two forms based on the type of tissue processing involved: IHC- formalin-fixed,paraffin-embedded (FFPE) and IHC-frozen (Fr). Often the preservation method is closely associated with the type of fixation. Formalin-fixed tissues are commonly paraffin-embedded following fixation, while frozen tissue sections can be fixed with formaldehyde or alcohol prior to or following cryosectioning.

7. IHC sample preparation Flowchart;

8. Fixatives used in immunohistochemistry; Standard fixatives; Aldehydes; 4% formaldehyde in phosphate-buffered saline (PBS) is the most common fixative for preserving protein targets in tissues. Formaldehyde reacts with amino groups in proteins to form methylene bridges that crosslink proteins in tissue sections. These molecular crosslinks can mask protein epitopes from antibody binding and may require the addition of an antigen retrieval step for proper IHC staining. Additionally, formaldehyde-mediated tissue fixation has been shown to induce translocation of phosphorylation-dependent epitopes from the membrane to the cytoplasm. Alcohols; The predominant alcohols used for fixation are ≥70% methanol and ≥80% ethanol. Alcohols work by removing and replacing water molecules in tissue, which can destabilize Hydrophobic bonds and alter the tertiary structure of proteins. This also causes the Precipitation of soluble proteins, making alcohol-mediated fixation more appropriate for detection of membrane bound proteins.

9. Fixatives used in immunohistochemistry Acetone; Acetone is also used as a strong dehydrant and precipitant, typically applied to sections of snap-frozen tissues. Acetone fixation is generally mild and may be followed by fixation with alcohols or formaldehyde. What Is difference between paraformaldehyde , Formaldehyde and formalin? Paraformaldehyde (PFA) is the polymerized form of formaldehyde and is not itself a fixing agent. Formaldehyde can be prepared by dissolving PFA in PBS using heat and sodium hydroxide (NaOH). Formalin refers to a saturated formaldehyde solution and some commercial formalin solutions include methanol as a stabilizer to prevent formaldehyde polymerization. A 10% formalin solution is equivalent to a 3.7% formaldehyde solution

10. Labels used in immunohistochemistry 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. 3,30diaminobenzidene 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,particularlysince the development of the alkaline phosphatase-anti-alkaline phosphatase (APAAP) method in 1984 by Cordell.

11. Labels used in immunohistochemistry Bacterial-derived B-D-galactosidase has also been used as a tracerColloidal metal labelsWhen 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. Fluorescent labels Radiolabels Chromogens; 3,3a-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? a-naphthol pyronin - red-purple

12. Buffer mediums;

13. Fixation technique in immunohistochemistry

14. Antibodies detection methods: Monoclonal antibody; A monoclonal antibody is an antibody made by cloning a unique white blood cell. All subsequent antibodies derived this way trace back to a unique parent cell. Monoclonal antibodies can have monovalent affinity, binding only to the same epitope. Polyclonal Antibody; Polyclonal antibodies (pAbs) are a complex mixture of several antibodies that are usually produced by different B-cell clones of an animal. These antibodies recognize and bind to many different epitopes of a single antigen and hence can form lattices with the antigens. Antibody detection : Frequently, IHC uses the indirect method of detection in which a secondary antibody, directed against the constant region of primary antibody, carries the label. The indirect method is more sensitive than using a directly labeled primary antibody because multiple labeled secondary antibodies can bind to a single primary antibody. After incubating the tissue sample with the appropriate labeled antibody, the antigen can be detected by IF or by a chromogenic reaction

15. 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. New direct technique; 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.

16. Continuous,,, Two step indirect technique; A labeled secondary antibody directed against the immunoglobulinof 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.

17. Unlabelled antibody methods; Peroxidase anti peroxidase 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 Anti alkaline phosphatase method /APAAP Principle same as those described for the PAP method except that the PAP complex is replaced with an APAAP complex.
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

18. Biotin/,Avidin procedure; A biotinylated enzyme (HRP or AP) is pre-incubated with free avidin to form large avidin–biotin–enzyme complexes. Typically, the avidin–biotinylated enzyme are mixed together in a specified ratio to prevent avidin saturation and incubated for about 15 minutes at room temperature to form the complex.

19. Epitope /Antigen retrieval methods ; Masked epitopes can be recovered with an antigen retrieval step, which works to promote epitope availability and enhance
immunogenicity (Figure 2). Proteolytic-Induced Epitope Retrieval (PIER) and Heat-Induced Epitope Retrieval (HIER) are two
of the most widely used antigen retrieval methods for FFPE tissue sections.

20. Epitope retrieval protocols;

21. Epitope retrieval protocols; Different antigen retrieval methods 1 . Microwave oven using EDTA (PH 8)or citrate buffer( PH 6.5) 2. Pressure cooker for gentle tissue at 10.3 kpa 3. Steamers 4.Water bath at 95 -98 degree Celsius is most optimum for antigen retrieval

22. 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. 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.

23. Immunohistochemistry in practice; 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.

24. Limitations of immunohistochemistry 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. Availibility 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. Antigen loss; The specificity of an antibody for particular antigen and its ability to react with that antigen require the preservation of antigen configuration

25. Applications of immunohistochemistry;