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Encyclopedia > Immunohistochemistry

Immunohistochemistry or IHC refers to the process of localizing proteins in cells of a tissue section exploiting the principle of antibodies binding specifically to antigens in biological tissues. It takes its name from the roots "immuno," in reference to antibodies used in the procedure, and "histo," meaning tissue. Immunohistochemical staining is widely used in the diagnosis and treatment of cancer. Specific molecular markers are characteristic of particular cancer types. IHC is also widely used in basic research to understand the distribution and localization of biomarkers in different parts of a tissue. Each antibody binds to a specific antigen; an interaction similar to a lock and key. ... An antigen is a molecule that stimulates an immune response. ... Biological tissue is a collection of interconnected cells that perform a similar function within an organism. ... Cancer is a class of diseases or disorders characterized by uncontrolled division of cells and the ability of these to spread, either by direct growth into adjacent tissue through invasion, or by implantation into distant sites by metastasis (where cancer cells are transported through the bloodstream or lymphatic system). ...


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 FITC, rhodamine, or Texas Red, (see immunofluorescence). The latter method is of great use in confocal laser scanning microscopy, which is highly sensitive and can also be used to visualise interactions between multiple proteins. Glutathione Peroxidase 1 A peroxidase (eg. ... Immunoperoxidase stains are used in the microscopic examination of tissues. ... Fluorescein isothiocyanate Fluorescein is a fluorophore commonly used in microscopy, in a type of dye laser as the gain medium, and in forensics and serology to detect latent blood stains. ... Rhodamine B Rhodamine 6G Rhodamine is a family of related chemical compounds, fluorone dyes. ... For the professional wrestler, see Mark Calaway For the food, see chili con carne. ... Immunofluorescence is the labeling of antibodies or antigens with fluorescent dyes. ... Confocal laser scanning microscopy (CLSM or LSCM) is a valuable tool for obtaining high resolution images and 3-D reconstructions. ... The neck of a guitar showing the first four frets. ...

Contents

Antibody types

The antibodies used for specific detection can be polyclonal or monoclonal. Monoclonal antibodies are generally considered to exhibit greater specificity. Polyclonal antibodies are made by injecting animals with peptide antigens, and then 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. Polyclonal antibodies are antibodies that are derived from different B-cell lines. ... Monoclonal antibodies (mAb) are antibodies that are identical because they were produced by one type of immune cell, all clones of a single parent cell. ...


Antibodies can also be 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 primary antibodies. Hence, secondary antibodies recognize immunoglobulins of a particular species and are conjugated to either biotin or a reporter enzyme such as alkaline phosphatase or horseradish peroxidase. Some secondary antibodies are conjugated to fluorescent agents, such as the Alexa-Fluor family, are also frequently used for detection of proteins in IHC procedures. Protein concentration is generally measured by densitometry analysis, where the intensity of staining correlates with the amount of the protein of interest. Vitamin H redirects here. ... Ribbon diagram of the enzyme TIM, surrounded by the space-filling model of the protein. ... Ball and stick model of alkaline phosphatase Alkaline phosphatase (ALP) (EC 3. ... Binomial name Armoracia rusticana P.G. Gaertn. ... Glutathione Peroxidase 1 A peroxidase (eg. ... Fluorescence induced by exposure to ultraviolet light in vials containing various sized cadmium selenide (CdSe) quantum dots. ...


Sample preparation

In the procedure, depending on the purpose and the thickness of the experimental sample, either thin (about 5 μm) slices are taken of the tissue of interest, or if the tissue is not very thick and is penetrable it is used whole. The slicing is usually accomplished through the use of a cryostat, and slices are mounted on slides. "Free-floating IHC" uses slices that are not mounted, these slices are normally produced using a vibrating microtome. A micrometre (American spelling: micrometer, symbol µm) is an SI unit of length. ... Cryocoolers are refrigerators used to reach cryogenic temperatures. ... A microtome is a mechanical instrument used to cut very thin slices for microscopic examination. ...


Direct and indirect IHC

The direct method of immunohistochemical staining uses one labelled antibody, which binds directly to the antigen being stained for.
The direct method of immunohistochemical staining uses one labelled antibody, which binds directly to the antigen being stained for.

There are two strategies used for the immmunohistochemical detection of antigens in tissue, the direct method and the indirect method.In both cases, the tissue is treated to rupture the membranes, usually by using a kind of detergent called Triton X-100. Image File history File links Immunohistochemicalstaining1. ... Image File history File links Immunohistochemicalstaining1. ... // The cell membrane (also called the plasma membrane or plasmalemma) is a semipermeable lipid bilayer common to all living cells. ... Triton X-100 is a nonionic detergent which has a hydrophilic polyethylene oxide group (on average it has 9. ...


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. This technique utilizes only one antibody and the procedure is therefore simple and rapid. However, it can suffer problems with sensitivity due to little signal amplification and is in less common use than indirect methods. ... Each antibody binds to a specific antigen; an interaction similar to a lock and key. ... Fluorescein isothiocyanate Fluorescein is a fluorophore commonly used in microscopy, in a type of dye laser as the gain medium, and in forensics and serology to detect latent blood stains. ... Antiserum is blood serum containing antibodies. ... An antigen is a molecule that stimulates an immune response. ... Each antibody binds to a specific antigen; an interaction similar to a lock and key. ...

The indirect method of immunohistochemical staining uses one antibody against the antigen being probed for, and a second, labelled, antibody against the first.
The indirect method of immunohistochemical staining uses one antibody against the antigen being probed for, and a second, labelled, antibody against the first.

The indirect method involves an unlabeled primary antibody (first layer) which reacts with tissue antigen, and a labeled secondary antibody (second layer) which reacts with the primary antibody. (The secondary antibody must be against the IgG of the animal species in which the primary antibody has been raised.) This method is more sensitive due to signal amplification through several secondary antibody reactions with different antigenic sites on the primary antibody. The second layer antibody can be labeled with a fluorescent dye or an enzyme. Image File history File links Immunohistochemicalstaining2. ... Image File history File links Immunohistochemicalstaining2. ... An antigen is a molecule that stimulates an immune response. ... The primary antibody (in purple) binds to an antigen (in green). ... Schematic of antibody binding to an antigen An antibody is a protein complex used by the immune system to identify and neutralize foreign objects like bacteria and viruses. ... Ribbon diagram of the enzyme TIM, surrounded by the space-filling model of the protein. ...


In a common procedure, a biotinylated secondary antibody is coupled with streptavidin-horseradish peroxidase. This is reacted with 3,3'-Diaminobenzidine (DAB) to produce a brown staining wherever primary and secondary antibodies are attached in a process known as DAB staining. The reaction can be enhanced using nickel, producing a deep purple/gray staining. Streptavidin (less commonly spelled streptavidine) is a tetrameric protein purified from Streptomyces avidinii that binds very tightly to the vitamin biotin with a dissociation constant (Kd) of ~ 10^(–15) M. This is one of the strongest biochemical interactions known, and is widely taken advantage of in scientific laboratories. ... This is a hazardous compound used in permanent immunohistochemical staining. ... General Name, Symbol, Number nickel, Ni, 28 Chemical series transition metals Group, Period, Block 10, 4, d Appearance lustrous, metallic and silvery with a gold tinge Standard atomic weight 58. ...


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 make custom labeled antibodies against every antigen of interest.


Diagnostic IHC markers

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. 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. carcinoma vs melanoma). Neuroscience is a field of study which deals with the structure, function, development, genetics, biochemistry, physiology, pharmacology and pathology of the nervous system. ... Picture of a western blot with 5 vertical lanes A western blot (a. ... The molecular mass of a substance (less accurately called molecular weight and abbreviated as MW) is the mass of one molecule of that substance, relative to the unified atomic mass unit u (equal to 1/12 the mass of one atom of carbon-12). ... Anatomic pathology is a medical specialty (a branch of pathology) that is concerned with the diagnosis of disease based on the gross, microscopic, and molecular examination of cells and tissues. ...

Carcinoembryonic antigen (CEA) is a glycoprotein involved in cell adhesion. ... Adenocarcinoma is a form of carcinoma that originates in glandular tissue. ... The introduction to this article provides insufficient context for those unfamiliar with the subject matter. ... Alpha-fetoprotein (AFP) is a protein that is normally only produced in the fetus during its development. ... Yolk sac tumours are members of the germ cell tumour group of ovarian neoplasms. ... Hepatocellular carcinoma (HCC, also called hepatoma) is a primary malignancy (cancer) of the liver. ... Endoscopic image of GIST in fundus of stomach, seen on retroflexion. ... Prostate specific antigen (PSA, also known as kallikrein III, seminin, semenogelase, γ-seminoprotein and P-30 antigen) is a protein manufactured almost exclusively by the prostate gland; PSA is produced for the ejaculate where it liquifies the semen and allows sperm to swim freely. ... Prostate cancer is a disease in which cancer develops in the prostate, a gland in the male reproductive system. ... Estrogens (or oestrogens) are a group of steroid compounds that function as the primary female sex hormone. ... Progesterone is a C-21 steroid hormone involved in the female menstrual cycle, pregnancy (supports gestation) and embryogenesis of humans and other species. ... B cells are lymphocytes that play a large role in the humoral immune response (as opposed to the cell-mediated immune response). ... Lymphoma is a general term for cancers that develop in the lymphatic system. ... CD20 is a non-glycosylated phosphoprotein expressed on the surface of all mature B-cells. ...

Immunotherapy

Many proteins shown to be highly upregulated in pathological states by immunohistochemistry are porential targets for therapies utilising monoclonal antibodies. Monoclonal antibody therapy is the use of monoclonal antibodies (or Mab) to specifically target cells. ... Monoclonal antibodies (mAb) are antibodies that are identical because they were produced by one type of immune cell, all clones of a single parent cell. ...


For example, Her-2/neu (also known as Erb-B2) is highly expressed in a variety of cancer cell types. As such, antibodies against Her-2/neu have been FDA approved for clinical treatment of cancer under the drug name Herceptin. HER2/neu (also known as ErbB-2) is a member of the epidermal growth factor receptor (EGFR) family and is notable for its role in the pathogenesis of breast cancer and as a target of treatment. ...


References

External links

can i really edit it? where is the validity here? Pathology (from Greek pathos, feeling, pain, suffering; and logos, study of; see also -ology) is the study of the processes underlying disease and other forms of illness, harmful abnormality, or dysfunction. ... The term disease refers to an abnormal condition of an organism that impairs function. ... Necrosis (in Greek Νεκρός = Death) is the name given to accidental death of cells and living tissue. ... An infection is the detrimental colonization of a host organism by a foreign species. ... In medicine, ischemia (Greek ισχαιμία, isch- is restriction, hema or haema is blood) is a restriction in blood supply, generally due to factors in the blood vessels, with resultant damage or dysfunction of tissue. ... An abscess on the skin, showing the redness and swelling characteristic of inflammation. ... Wound healing, or wound repair, is the bodys natural process of regenerating dermal and epidermal tissue. ... Neoplasia (new growth in Greek) is abnormal, disorganized growth in a tissue or organ, usually forming a distinct mass. ... Anatomic pathology is a medical specialty (a branch of pathology) that is concerned with the diagnosis of disease based on the gross, microscopic, and molecular examination of cells and tissues. ... Anatomic pathology is a medical specialty (a branch of pathology) that is concerned with the diagnosis of disease based on the gross, microscopic, and molecular examination of cells and tissues. ... Cytopathology is a branch of pathology that studies and diagnoses diseases on the cellular level. ... Post-mortem, postmortem and post mortem redirect here. ... Molecular pathology is an emerging discipline within anatomic pathology which is focused on the use of nucleic acid-based techniques such as DNA sequencing, fluorescent in-situ hybridization, reverse-transcriptase polymerase chain reaction, and nucleic acid microarrays for specialised studies of disease in tissues and cells. ... Forensic pathology, first recognized in the USA by the American Board of Pathology in 1959, is a branch of medicine concerned with determining cause of death usually for civil or criminal law cases. ... Dental pathologists are doctors of dental science who specialise in the diagnosis and characterization of diseases of the teeth, jaw, and maxilla through the examination of tissue specimens. ... Gross examination or grossing is the process by which pathology specimens are inspected with the naked eye to obtain diagnostic information, while being processed for further microscopic examination. ... Histopathology is a field of pathology which specialises in the histologic study of diseased tissue. ... The electron microscope is a microscope that can magnify very small details with high resolving power due to the use of electrons rather than light to scatter off material, magnifying at levels up to 500,000 times. ... Immunofluorescence is the labeling of antibodies or antigens with fluorescent dyes. ... A metaphase cell positive for the bcr/abl rearrangement using FISH. The chromosomes can be seen in blue. ... Clinical Pathology is one of the two major divisions of Pathology. ... Chemical pathology (also known as clinical biochemistry or clinical chemistry) is the area of pathology that is generally concerned with analysis of bodily fluids. ... Hematopathology is the branch of pathology which studies diseases of hematopoietic cells (see below). ... Transfusion medicine (or transfusiology) is the branch of medicine that is concerned with the transfusion of blood and blood components. ... Medical microbiology is a branch of microbiology which deals with the study of microorganisms including bacteria, viruses, fungi and parasites which are of medical importance and are capable of causing diseases in human beings. ... The specificity of the bond between antibody and antigen has made it an excellent tool in the detection of substances in a variety of diagnostic techniques, known collectively as diagnostic immunology. ... Enzyme assays are laboratory methods for measuring enzymatic activity. ... Mass spectrometry (also known as mass spectroscopy (deprecated)[1] or informally, mass-spec and MS) is an analytical technique used to measure the mass-to-charge ratio of ions. ... Pictured is a sophisticated gas chromatography system. ... Flow cytometry is a technique for counting, examining and sorting microscopic particles suspended in a stream of fluid. ... A blood bank is a cache or bank of blood or blood components, gathered as a result of blood donation, stored and preserved for later use in blood transfusions. ... A microbiological culture is a way to determine the cause of infectious disease by letting the agent multiply (reproduce) in predetermined media. ... Serology is literally the scientific study of the blood serum. ...


  Results from FactBites:
 
Immunohistochemistry (365 words)
Immunohistochemistry is an important application of monoclonal as well as polyclonal antibodies to determine the tissue distribution of an antigen of interest in health and disease.
Immunohistochemistry is widely used for diagnosis of cancers, because specific tumor antigens are expressed de novo or upregulated in certain cancers.
Immunohistochemistry requires the availability of biopsies, these are processed into sections with a microtome, then the sections are incubated with an appropriate antibody.
IMMUNOHISTOCHEMISTRY IN CNS (1972 words)
Immunohistochemistry is an amalgamation of immunology and histology.
The vast progress in the field of immunohistochemistry along with the knowledge of cell and molecular biology allows the exploration of the molecular phenotypes of the developing CNS tumors.
The future of immunohistochemistry is aimed at not only the diagnosis and prognostication of the tumors but also being able to comment upon the probable response to various chemotherapeutic agents.
  More results at FactBites »

 
 

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