Mouse-brain slice stained by Immunohistochemistry.
Immunohistochemistry or IHC refers to the process of detecting antigens (e.g., proteins) in cells of a tissue section by exploiting the principle of antibodies binding specifically to antigens in biological tissues.[1] IHC takes its name from the roots "immuno," in reference to antibodies used in the procedure, and "histo," meaning tissue (compare to immunocytochemistry). The procedure was conceptualized and first implemented by Dr. Albert Coons in 1941.[2]
Immunohistochemical staining is widely used in the diagnosis of abnormal cells such as those found in cancerous tumors. A video showing preparation for immunohistochemistry (8 minutes) and then 20 minutes scanning through immunohistochemically stained tissues near colon cancers, shows different degrees of deficiency in three DNA repair proteins (ERCC1, PMS2 and KU86) and one mitochondrial protein (CCOI).[3] Specific molecular markers are characteristic of particular cellular events such as proliferation or cell death (apoptosis). IHC is also widely used in basic research to understand the distribution and localization of biomarkers and differentially expressed proteins in different parts of a biological tissue.
Visualising an antibody-antigen interaction can be accomplished in a number of ways. In the most common instance, an antibody is conjugated to an enzyme, such as peroxidase, that can catalyse a colour-producing reaction (see immunoperoxidase staining). Alternatively, the antibody can also be tagged to a fluorophore, such as fluorescein or rhodamine (see immunofluorescence).
Contents
- 1 Sample preparation
- 2 Sample labeling
- 2.1 Antibody types
- 2.2 IHC reporters
- 2.3 Target antigen detection methods
- 2.4 Counterstains
- 3 IHC troubleshooting
- 4 Diagnostic IHC markers
- 5 Directing therapy
- 5.1 Chemical inhibitors
- 5.2 Monoclonal antibodies
- 6 See also
- 7 References
- 8 External links
Sample preparation[edit]
Preparation of the sample is critical to maintain cell morphology, tissue architecture and the antigenicity of target epitopes. This requires proper tissue collection, fixation and sectioning. A solution of paraformaldehyde is often used to fixate tissue, but other methods may be used. The tissue may then be sliced or used whole, dependant upon the purpose of the experiment or the tissue itself. Sections can be sliced on a variety of instruments, most commonly a microtome or cryostat and are sliced at a range of 4-40 μm.[citation needed] Before sectioning, the tissue sample may be embedded in a medium, like paraffin wax or cryomedia. Slices may then be mounted on slides for visualizaton through a microscope.
Unlike immunocytochemistry, the tissue does not need to be permeabilized because this has already been accomplished by the microtome blade during sample preparation. Detergents like Triton X-100 are generally used in immunohistochemistry to reduce surface tension, allowing less reagent to be used to achieve better and more even coverage of the sample.
Dependent on the method of fixation and tissue preservation, the sample may require additional steps to make the epitopes available for antibody binding, including deparaffinization and antigen retrieval (microwave method, enzyme method, hot incubation method.) These steps may make the difference between the target antigens staining or not staining.
Dependent on the tissue type and the method of antigen detection, endogenous biotin or enzymes may need to be blocked or quenched, respectively, prior to antibody staining. Although antibodies show preferential avidity for specific epitopes, they may partially or weakly bind to sites on nonspecific proteins (also called reactive sites) that are similar to the cognate binding sites on the target antigen. A great amount of non-specific binding causes high background staining which will mask the detection of the target antigen. To reduce background staining in IHC, ICC and other immunostaining methods, samples are incubated with a buffer that blocks the reactive sites to which the primary or secondary antibodies may otherwise bind. Common blocking buffers include normal serum, non-fat dry milk, BSA, or gelatin. Commercial blocking buffers with proprietary formulations are available for greater efficiency.
Sample labeling[edit]
Antibody types[edit]
The antibodies used for specific detection can be polyclonal or monoclonal. Polyclonal antibodies are made by injecting animals with peptide Ag and, after a secondary immune response is stimulated, isolating antibodies from whole serum. Thus, polyclonal antibodies are a heterogeneous mix of antibodies that recognize several epitopes. Monoclonal antibodies show specificity for a single epitope and are therefore considered more specific to the target antigen than polyclonal antibodies.
For IHC detection strategies, antibodies are classified as primary or secondary reagents. Primary antibodies are raised against an antigen of interest and are typically unconjugated (unlabelled), while secondary antibodies are raised against immunoglobulins of the primary antibody species. The secondary antibody is usually conjugated to a linker molecule, such as biotin, that then recruits reporter molecules, or the secondary antibody itself is directly bound to the reporter molecule.
IHC reporters[edit]
Reporter molecules vary based on the nature of the detection method, the most popular being chromogenic and fluorescence detection mediated by an enzyme or a fluorophore, respectively. With chromogenic reporters, an enzyme label is reacted with a substrate to yield an intensely colored product that can be analyzed with an ordinary light microscope. While the list of enzyme substrates is extensive, Alkaline phosphatase (AP) and horseradish peroxidase (HRP) are the two enzymes used most extensively as labels for protein detection. An array of chromogenic, fluorogenic and chemiluminescent substrates is available for use with either enzyme, including DAB or BCIP/NBT, which produce a brown or purple staining, respectively, wherever the enzymes are bound.
Reaction with DAB can be enhanced using nickel[citation needed], producing a deep purple/black staining. Fluorescent reporters are small, organic molecules used for IHC detection and traditionally include FITC, TRITC and AMCA, while commercial derivatives, including the Alexa Fluors and Dylight Fluors, show similar enhanced performance but vary in price. For chromogenic and fluorescent detection methods, densitometric analysis of the signal can provide semi- and fully quantitative data, respectively, to correlate the level of reporter signal to the level of protein expression or localization.
The direct method of immunohistochemical staining uses one labelled antibody, which binds directly to the antigen being stained for.
The
indirect method of immunohistochemical staining uses one antibody against the antigen being probed for, and a second, labelled, antibody against the first.
Target antigen detection methods[edit]
The direct method is a one-step staining method and involves a labeled antibody (e.g. FITC-conjugated antiserum) reacting directly with the antigen in tissue sections. While this technique utilizes only one antibody and therefore is simple and rapid, the sensitivity is lower due to little signal amplification, in contrast to indirect approaches. Ergo, this stratagem is utilized with lesser frequency than its multi-phase counterpart.
The indirect method involves an unlabeled primary antibody (first layer) that binds to the target antigen in the tissue and a labeled secondary antibody (second layer) that reacts with the primary antibody. As mentioned above, the secondary antibody must be raised against the IgG of the animal species in which the primary antibody has been raised. This method is more sensitive than direct detection strategies because of signal amplification due to the binding of several secondary antibodies to each primary antibody if the secondary antibody is conjugated to the fluorescent or enzyme reporter.
Further amplification can be achieved if the secondary antibody is conjugated to several biotin molecules, which can recruit complexes of avidin-, streptavidin or NeutrAvidin proteinbound-enzyme. The difference between these three biotin-binding proteins is their individual binding affinity to endogenous tissue targets leading to nonspecific binding and high background; the ranking of these proteins based on their nonspecific binding affinities, from highest to lowest, is: 1) avidin, 2) streptavidin and 3) Neutravidin protein.
The indirect method, aside from its greater sensitivity, also has the advantage that only a relatively small number of standard conjugated (labeled) secondary antibodies needs to be generated. For example, a labeled secondary antibody raised against rabbit IgG, which can be purchased "off the shelf," is useful with any primary antibody raised in rabbit. With the direct method, it would be necessary to label each primary antibody for every antigen of interest.
Counterstains[edit]
After immunohistochemical staining of the target antigen, a second stain is often applied to provide contrast that helps the primary stain stand out. Many of these stains show specificity for discrete cellular compartments or antigens, while others will stain the whole cell. Both chromogenic and fluorescent dyes are available for IHC to provide a vast array of reagents to fit every experimental design, and include: hematoxylin, Hoechst stain and DAPI are commonly used.
IHC troubleshooting[edit]
In immunohistochemical techniques, there are several steps prior to the final staining of the tissue antigen, and many potential problems affect the outcome of the procedure. The major problem areas in IHC staining include strong background staining, weak target antigen staining and autofluorescence. Endogenous biotin or reporter enzymes or primary/secondary antibody cross-reactivity are common causes of strong background staining, while weak staining may be caused by poor enzyme activity or primary antibody potency. Furthermore, autofluorescence may be due to the nature of the tissue or the fixation method. These aspects of IHC tissue prep and antibody staining must be systematically addressed to identify and overcome staining issues.
Diagnostic IHC markers[edit]
Immunohistochemical staining of normal kidney with CD10.
IHC is an excellent detection technique and has the tremendous advantage of being able to show exactly where a given protein is located within the tissue examined. It is also an effective way to examine the tissues .This has made it a widely used technique in the neurosciences, enabling researchers to examine protein expression within specific brain structures. Its major disadvantage is that, unlike immunoblotting techniques where staining is checked against a molecular weight ladder, it is impossible to show in IHC that the staining corresponds with the protein of interest. For this reason, primary antibodies must be well-validated in a Western Blot or similar procedure. The technique is even more widely used in diagnostic surgical pathology for typing tumors (e.g. immunostaining for e-cadherin to differentiate between DCIS (ductal carcinoma in situ: stains positive) and LCIS (lobular carcinoma in situ: does not stain positive)[4]).
- Carcinoembryonic antigen (CEA): used for identification of adenocarcinomas. Not specific for site.
- Cytokeratins: used for identification of carcinomas but may also be expressed in some sarcomas.[5]
- CD15 and CD30 : used for Hodgkin's disease
- Alpha fetoprotein: for yolk sac tumors and hepatocellular carcinoma
- CD117 (KIT): for gastrointestinal stromal tumors (GIST)
- CD10 (CALLA): for renal cell carcinoma and acute lymphoblastic leukemia
- Prostate specific antigen (PSA): for prostate cancer
- estrogens and progesterone staining for tumour identification
- Identification of B-cell lymphomas using CD20
- Identification of T-cell lymphomas using CD3
Directing therapy[edit]
A variety of molecular pathways are altered in cancer and some of the alterations can be targeted in cancer therapy. Immunohistochemistry can be used to assess which tumors are likely to respond to therapy, by detecting the presence or elevated levels of the molecular target.
Chemical inhibitors[edit]
Tumor biology allows for a number of potential intracellular targets. Many tumors are hormone dependent. The presence of hormone receptors can be used to determine if a tumor is potentially responsive to antihormonal therapy. One of the first therapies was the antiestrogen, tamoxifen, used to treat breast cancer. Such hormone receptors can be detected by immunohistochemistry.[6] Imatinib, an intracellualar tyrosine kinase inhibitor, was developed to treat chronic myelogenous leukemia, a disease characterized by the formation of a specific abnormal tyrosine kinase. Imitanib has proven effective in tumors that express other tyrosine kinases, most notably KIT. Most gastrointestinal stromal tumors express KIT, which can be detected by immunohistochemistry.[7]
Monoclonal antibodies[edit]
Main article: Monoclonal antibody therapy
Many proteins shown to be highly upregulated in pathological states by immunohistochemistry are potential targets for therapies utilising monoclonal antibodies. Monoclonal antibodies, due to their size, are utilized against cell surface targets. Among the overexpressed targets are members of the epidermal growth factor receptor (EGFR) family, transmembrane proteins with an extracellular receptor domain regulating an intracellular tyrosine kinase.[8] Of these, HER2/neu (also known as Erb-B2) was the first to be developed. The molecule is highly expressed in a variety of cancer cell types, most notably breast cancer. As such, antibodies against HER2/neu have been FDA approved for clinical treatment of cancer under the drug name Herceptin. There are commercially available immunohistochemical tests, Dako HercepTest,[9] Leica Biosystems Oracle[10] and Ventana Pathway.[11]
Similarly, EGFR (HER-1) is overexpressed in a variety of cancers including head and neck and colon. Immunohistochemistry is used to determine patients who may benefit from therapeutic antibodies such as Erbitux (cetuximab).[12] Commercial systems to detect EGFR by immunohistochemistry include the Dako pharmDx.[13]
See also[edit]
- Cutaneous conditions with immunofluorescence findings
References[edit]
- ^ Ramos-Vara, JA (2005). "Technical Aspects of Immunohistochemistry". Vet Pathol 42 (4): 405–426. doi:10.1354/vp.42-4-405. PMID 16006601.
- ^ Coons AH,Creech HJ, Jones RN: Immunological properties of an antibody containing a fluorescent group. Proc Soc Exp Biol Med 1941; 47: 200-202.
- ^ Nguyen H, Loustaunau C, Facista A, Ramsey L, Hassounah N, Taylor H, Krouse R, Payne CM, Tsikitis VL, Goldschmid S, Banerjee B, Perini RF, Bernstein C. (2010). Deficient Pms2, ERCC1, Ku86, CcOI in field defects during progression to colon cancer. J Vis Exp (41). doi:pii: 1931. 10.3791/1931. PMID 20689513 Best seen by selecting “Free in PMC” and then clicking on “Download video file.(333M, mp4)” located just under the first image. This allows the viewer to download the entire 28 minute video. The downloaded video allows the viewer to fast-forward or to pause and go back to review sections of special interest.
- ^ O'Malley F and Pinder S, Breast Pathology, 1st. Ed. Elsevier 2006. ISBN 978-0-443-06680-1
- ^ Leader M, Patel J, Makin C, Henry K (December 1986). "An analysis of the sensitivity and specificity of the cytokeratin marker CAM 5.2 for epithelial tumours. Results of a study of 203 sarcomas, 50 carcinomas and 28 malignant melanomas". Histopathology 10 (12): 1315–24. doi:10.1111/j.1365-2559.1986.tb02574.x. PMID 2434403.
- ^ Jørgensen, Jan Trøst; Kirsten Vang Nielsen, Bent Ejlertsen (April 2007). "Pharmacodiagnostics and targeted therapies - a rational approach for individualizing medical anticancer therapy in breast cancer". The Oncologist (United States: AlphaMed Press) 12 (4): 397–405. doi:10.1634/theoncologist.12-4-397. ISSN 1083-7159. PMID 17470682. Retrieved 2008-03-14.
- ^ Gold JS, Dematteo RP (August 2006). "Combined Surgical and Molecular Therapy: The Gastrointestinal Stromal Tumor Model". Ann. Surg. 244 (2): 176–84. doi:10.1097/01.sla.0000218080.94145.cf. PMC 1602162. PMID 16858179.
- ^ Harari, P M (December 2004). "Epidermal growth factor receptor inhibition strategies in oncology". Endocrine-Related Cancer (England: Society for Endocrinology) 11 (4): 689–708. doi:10.1677/erc.1.00600. ISSN 1351-0088. PMID 15613446. Retrieved 2008-03-14.
- ^ http://www.dakousa.com/index/prod_search/prod_baseproducts.htm?productareaid=1&productgroupid=3&productsubgroupid=1003000
- ^ "leicabiosystems.com". leicabiosystems.com. Retrieved 2013-06-16.
- ^ Press, Michael F.; Guido Sauter, Leslie Bernstein, Ivonne E.Villalobos, MartinaMirlacher, Jian-Yuan Zhou, RoobaWardeh, Yong-Tian Li, Roberta Guzman, Yanling Ma, Jane Sullivan-Halley, Angela Santiago, Jinha M. Park, Alessandro Riva, Dennis J.Slamon (September 15, 2005). "Diagnostic evaluation of HER-2 as a molecular target: an assessment of accuracy and reproducibility of laboratory testing in large, prospective, randomized clinical trials". Clinical Cancer Research (United States: American Association for Cancer Research.). 2005 15;11(18): (18): 6598–6607. doi:10.1158/1078-0432.CCR-05-0636. ISSN 1078-0432. PMID 16166438. Retrieved 2008-03-14.
- ^ Bibeau F, Boissière-Michot F, Sabourin JC, et al. (September 2006). "Assessment of epidermal growth factor receptor (EGFR) expression in primary colorectal carcinomas and their related metastases on tissue sections and tissue microarray". Virchows Arch. 449 (3): 281–7. doi:10.1007/s00428-006-0247-9. PMC 1888717. PMID 16865406.
- ^ http://www.dakousa.com/index/prod_search/prod_groups.htm?productareaid=1
External links[edit]
- Overview of Immunohistochemistry--describes all aspects of IHC including sample prep, staining and troubleshooting
- Yale Core Tissue Microarray Facility
- Histochemical Staining Methods - University of Rochester Department of Pathology
- Immunohistochemistry Staining Protocol
- Burnett R, Guichard Y, Barale E (1997). "Immunohistochemistry for light microscopy in safety evaluation of therapeutic agents: an overview". Toxicology 119 (1): 83–93. doi:10.1016/S0300-483X(96)03600-1. PMID 9129199.
- custom immunohistochemistry(IHC)detection methods
- Immunohistochemistry at the US National Library of Medicine Medical Subject Headings (MeSH)
- Joyner, A.; Wall, N. (2008). "Immunohistochemistry of Whole-Mount Mouse Embryos". Cold Spring Harbor Protocols 2008 (2): pdb.prot4820. doi:10.1101/pdb.prot4820.
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Proteins: key methods of study
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| Experimental |
- Protein purification
- Green fluorescent protein
- Western blot
- Protein immunostaining
- Protein sequencing
- Gel electrophoresis/Protein electrophoresis
- Protein immunoprecipitation
- Peptide mass fingerprinting
- Dual polarization interferometry
- Microscale thermophoresis
- Chromatin immunoprecipitation
- Surface Plasmon Resonance
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| Bioinformatics |
- Protein structure prediction
- Protein–protein docking
- Protein structural alignment
- Protein ontology
- Protein–protein interaction prediction
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| Assay |
- Enzyme assay
- Protein assay
- Secretion assay
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| Display techniques |
- Bacterial display
- mRNA display
- Phage display
- Ribosome display
- Yeast display
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| Super resolution microscopy |
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Medicine: Pathology
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| Principles of pathology |
- Disease
- Hemodynamics
- Inflammation
- Wound healing
- Cell death
- Necrosis
- Liquefactive necrosis
- Coagulative necrosis
- Caseous necrosis
- Fat necrosis
- Apoptosis
- Pyknosis
- Karyorrhexis
- Karyolysis
- Cellular adaptation
- Atrophy
- Hypertrophy
- Hyperplasia
- Dysplasia
- Metaplasia
- Squamous
- Glandular
- Accumulations:
- pigment
- Hemosiderin
- Lipochrome/Lipofuscin
- Melanin
- Steatosis
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| Anatomical pathology |
- Surgical pathology
- Cytopathology
- Autopsy
- Molecular pathology
- Forensic pathology
- Dental pathology
- Gross examination
- Histopathology
- Immunohistochemistry
- Electron microscopy
- Immunofluorescence
- Fluorescent in situ hybridization
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| Clinical pathology |
- Clinical chemistry
- Hematopathology
- Transfusion medicine
- Medical microbiology
- Diagnostic immunology
- Immunopathology
- Enzyme assay
- Mass spectrometry
- Chromatography
- Flow cytometry
- Blood bank
- Microbiological culture
- Serology
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| Specific conditions |
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Medical test: Immunologic techniques and tests (CPT 86000–86849)
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Immunologic techniques
and tests ·
serology/
diagnostic immunology |
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Immunoprecipitation
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Chromatin immunoprecipitation · Immunodiffusion (Ouchterlony double immunodiffusion, Radial immunodiffusion, Immunoelectrophoresis, Counterimmunoelectrophoresis)
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Immunoassay
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ELISA · ELISPOT · Enzyme Multiplied Immunoassay Technique · RAST test · Radioimmunoassay · Radiobinding assay · Immunofluorescence
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Agglutination
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Hemagglutination/Hemagglutinin (Coombs test) · Latex fixation test
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Other
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Nephelometry · Complement fixation test · Immunocytochemistry · Immunohistochemistry (Direct fluorescent antibody) · Epitope mapping · Skin allergy test · Patch test
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| Inflammation |
C-reactive protein · Procalcitonin
Total complement activity · MELISA
CBC (lymphocyte count)
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cell/phys/auag/auab/comp, igrc
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