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Encyclopedia > Immunostaining

Immunostaining is a general term in biochemistry that applies to any use of an antibody-based method to detect a specific protein in a sample. The term immunostaining was originally used to refer to the immunohistochemical staining of tissue sections, as first described by Albert Coons in 1941.[1] Now however, immunostaining encompasses a broad range of techniques used in histology, cell biology, and molecular biology that utilise antibody-based staining methods. Biochemistry is the study of the chemical processes and transformations in living organisms. ... Each antibody binds to a specific antigen; an interaction similar to a lock and key. ... A representation of the 3D structure of myoglobin, showing coloured alpha helices. ... Immunohistochemical staining is a common immunological technique used in the biological sciences for the detection of proteins within the context of the tissue in which the protein is found. ... A thin section of lung tissue stained with hematoxylin and eosin. ... This article or section includes a list of works cited or a list of external links, but its sources remain unclear because it lacks in-text citations. ... Molecular biology is the study of biology at a molecular level. ...

Contents

Immunostaining Techniques

Immunohistochemistry

Main article: Immunohistochemistry

Immunohistochemistry or IHC staining of cells or tissue sections is perhaps the most commonly applied immunostaining technique. [2] While the first cases of IHC staining used fluorescent dyes (see immunofluorescence), other non-fluorescent methods using enzymes such as peroxidase (see immunoperoxidase staining) and alkaline phosphatase are now used. These enzymes are capable of catalysing reactions that give a coloured product that is easily detectable by light microscopy. Alternatively, radioactive elements can be used as labels, and the immunoreaction can be visualized by autoradiography.[3] 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. ... Drawing of the structure of cork as it appeared under the microscope to Robert Hooke from Micrographia which is the origin of the word cell being used to describe the smallest unit of a living organism Cells in culture, stained for keratin (red) and DNA (green) The cell is the... Biological tissue is a collection of interconnected cells that perform a similar function within an organism. ... Fluorescence induced by exposure to ultraviolet light in vials containing various sized cadmium selenide (CdSe) quantum dots. ... Look up dye in Wiktionary, the free dictionary. ... Immunofluorescence is the labeling of antibodies or antigens with fluorescent dyes. ... Ribbon diagram of the enzyme TIM, surrounded by the space-filling model of the protein. ... Glutathione Peroxidase 1 A peroxidase (eg. ... Immunoperoxidase stains are used in the microscopic examination of tissues. ... Ball and stick model of alkaline phosphatase Alkaline phosphatase (ALP) (EC 3. ... This article or section is not written in the formal tone expected of an encyclopedia article. ... Radioactive decay is the set of various processes by which unstable atomic nuclei (nuclides) emit subatomic particles. ... The periodic table of the chemical elements A chemical element, or element, is a type of atom that is defined by its atomic number; that is, by the number of protons in its nucleus. ... An autoradiograph is an image produced on a photographic film by the radiation from a radioactive substance. ...


Tissue preparation or fixation is essential for the preservation of cell morphology and tissue architecture. Inappropriate or prolonged fixation may significantly diminish the antibody binding capability. Many antigens can be successfully demonstrated in formalin-fixed paraffin-embedded tissue sections. However, some antigens will not survive even moderate amounts of aldehyde fixation. Under these conditions, tissues should be rapidly fresh frozen in liquid nitrogen and cut with a cryostat. The disadvantages of frozen sections include poor morphology, poor resolution at higher magnifications, difficulty in cutting over paraffin sections, and the need for frozen storage. Alternatively, vibratome sections do not require the tissue to be processed through organic solvents or high heat, which can destroy the antigenicity, or disrupted by freeze thawing. The disadvantage of vibratome sections is that the sectioning process is slow and difficult with soft and poorly fixed tissues, and that chatter marks or vibratome lines are often apparent in the sections. The chemical compound formaldehyde (also known by IUPAC nomenclature as methanal), is a gas with a strong pungent smell. ... This article or section is in need of attention from an expert on the subject. ... General Name, Symbol, Number nitrogen, N, 7 Chemical series nonmetals Group, Period, Block 15, 2, p Appearance colorless gas Standard atomic weight 14. ...


The detection of many antigens can be dramatically improved by antigen retrieval methods that act by breaking some of the protein cross-links formed by fixation to uncover hidden antigenic sites. This can be accomplished by heating for varying lengths of times (heat induced epitope retrieval or HIER) or using enzyme digestion (proteolytic induced epitope retrieval or PIER).


One of the main difficulties with IHC staining is overcoming specific or non-specific background. Optimisation of fixation methods and times, pre-treatment with blocking agents, incubating antibodies with high salt, and optimising post-antibody wash buffers and wash times are all important for obtaining high quality immunostaining. In addition, the presence of positive controls and negative controls for staining are essential for determining specificity. Please wikify (format) this article as suggested in the Guide to layout and the Manual of Style. ... A scientific control augments integrity in experiments by isolating variables as dictated by the scientific method in order to make a conclusion about such variables. ...


Flow cytometry

Main article: flow cytometry

A flow cytometer can be used for the direct analysis of cells expressing one or more specific proteins. Cells are immunostained in solution using methods similar to used for immunofluorescence, and then analysed by flow cytometry. Analysis of a marine sample of photosynthetic picoplankton by flow cytometry showing three different populations (Prochlorococcus, Synechococcus and picoeukaryotes) Flow cytometry is a technique for counting, examining and sorting microscopic particles suspended in a stream of fluid. ... Flow cytometry is a technique for counting, examining and sorting microscopic particles suspended in a stream of fluid. ...


Flow cytometry has several advantages over IHC including: the ability to define distinct cell populations are defined by their size and granularity; the capacity to gate out dead cells; improved sensitivity; and multi-colour analysis to measure several antigens simultaneously. However, flow cytometry can be less effective at detecting extremely rare cell populations, and there is a loss of architectural relationships in the absence of a tissue section.[4] Flow cytometry also has a high capital cost associated with the purchase of a flow cytometer.


Western blotting

Main article: western blot

Western blotting allows the detection of specific proteins from extracts made from cells or tissues, before or after any purification steps. Proteins are generally separated by size using gel electrophoresis before being transferred to a synthetic membrane via dry, semi-dry, or wet blotting methods. The membrane can then be probed using antibodies using methods similar to immunohistochemistry, but without a need for fixation. Detection is typically performed using peroxidase linked antibodies to catalyse a chemiluminescent reaction. Figure 1. ... Protein purification is the process of isolating proteins from a homogenate, which may comprise cell and tissue components, including DNA, cell membrane and other proteins. ... Gel electrophoresis is the separation of deoxyribonucleic acid, ribonucleic acid, or protein through an electric charge. ... In chemistry, chemical synthesis is purposeful execution of chemical reactions in order to get a product, or several products. ... An artificial membrane, also called a synthetic membrane, is a membrane prepared for separation tasks in laboratory and industry. ... Glutathione Peroxidase 1 A peroxidase (eg. ... A chemoluminescent reaction carried out in an erlenmeyer flask producing a large amount of light. ...


Western blotting is a routine molecular biology method that can be used to semi-quantitatively compare protein levels between extracts. The size separation prior to blotting allows the protein molecular weight to be gauged as compared with known molecular weight markers. 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). ...


Enzyme-linked immunosorbent assay

Main article: ELISA

The enzyme-linked immunosorbent assay or ELISA is a diagnostic method for quantitatively or semi-quantitatively determining protein concentrations from blood plasma, serum or cell/tissue extracts in a multi-well plate format (usually 96-wells per plate). Broadly, proteins in solution are adsorbed to ELISA plates. Antibodies specific for the protein of interest are used to probe the plate. Background is minimised by optimising blocking and washing methods (as for IHC), and specificity is ensured via the presence of positive and negative controls. Detection methods are usually colorimetric or chemiluminescence based. Elisa (born Elisa Toffoli on 19 December 1977) is an Italian singer and solo artist, writing and performing within several genres, notably rock, blues, soul and ambient. ... Blood plasma is the liquid component of blood, in which the blood cells are suspended. ... Look up Serum in Wiktionary, the free dictionary. ...


Immuno-electron microscopy

Electron microscopy or EM can be used to study the detailed microarchitecture of tissues or cells. Immuno-EM allows the detection of specific proteins in ultrathin tissue sections. Antibodies labelled with heavy metal particles (e.g. gold) can be directly visualised using transmission electron microscopy. While powerful in detecting the sub-cellular localisation of a protein, immuno-EM can be technically challenging, expensive, and require rigorous optimisation of tissue fixation and processing methods. 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. ... A section of a cell of Bacillus subtilis, taken with a Tecnai T-12 TEM. The scale bar is 200nm. ...


Methodological overview

In immunostaining methods, an antibody is used to detect a specific protein epitope. These antibodies can be monoclonal or polyclonal. Detection of this first or primary antibody can be accomplished in multiple ways. Wikipedia does not yet have an article with this exact name. ... A representation of the 3D structure of myoglobin, showing coloured alpha helices. ... An epitope is the part of a macromolecule that is recognized by the immune system, specifically by antibodies, B cells, or cytotoxic T 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. ... Polyclonal antibodies are antibodies that are derived from different B-cell lines. ...

  • The primary antibody can be directly labeled using an enzyme or fluorophore.
  • The primary antibody can be labeled using a small molecule which interacts with a high affinity binding partner that can be linked to an enzyme or fluorophore. The biotin-strepavidin is one commonly used high affinity interaction.
  • The primary antibody can be probed for using a broader species-specific secondary antibody that is labeled using an enzyme, or fluorophore.
  • In the case of electron microscopy, antibodies are linked to a heavy metal atom.

As previously described, enzymes such as horseradish peroxidase or alkaline phosphatase are commonly used to catalyse reactions that give a coloured or chemiluminescent product. Fluorescent molecules can be visualised using fluoresence microscopy or confocal microscopy. Ribbon diagram of the enzyme TIM, surrounded by the space-filling model of the protein. ... A fluorophore is a component of a molecule which causes a molecule to be fluorescent. ... Vitamin H redirects here. ... The primary antibody (in purple) binds to an antigen (in green). ... An electron microscope is a type of microscope that uses electrons to illuminate and create an image of a specimen. ... For other uses, see Atom (disambiguation). ... Binomial name Armoracia rusticana P.G. Gaertn. ... Glutathione Peroxidase 1 A peroxidase (eg. ... Ball and stick model of alkaline phosphatase Alkaline phosphatase (ALP) (EC 3. ... Lightsticks Chemoluminescence (sometimes chemiluminescence) is the emission of light (luminescence) as the result of a chemical reaction. ... Fluorescence induced by exposure to ultraviolet light in vials containing various sized cadmium selenide (CdSe) quantum dots. ... Confocal microscopy is an imaging technique used to increase micrograph contrast and/or to reconstruct three-dimensional images by using a spatial pinhole to eliminate out-of-focus light or flare in specimens that are thicker than the focal plane. ...


Applications of Immunostaining

The applications of immunostaining are numerous, but are most typically used in clinical diagnostics and laboratory research.


Clinically, IHC is used in histopathology for the diagnosis of specific types of cancers based on molecular markers. Histopathology is a field of pathology which specialises in the histologic study of diseased tissue. ...


In laboratory science, immunostaining can be used for a variety of applications based on investigating the presence or absence of a protein, its tissue distribution, its sub-cellular localisation, and or changes in protein expression or degradation.


References

  1. ^ Coons, Albert (1941). "Immunological properties of an antibody containing a fluorescent group". Proc Soc Exp Biol Med 47: 200-202. 
  2. ^ Ramos-Vara, JA (2005). "Techical Aspects of Immunohistochemistry". Vet Pathol 42: 405-426. 
  3. ^ http://www.ihcworld.com/introduction.htm
  4. ^ Cherie, H (2004). "Applications of Flow Cytometry and Immunohistochemistry to Diagnostic Hematopathology". Archives of Pathology and Laboratory Medicine 128 (9): 1004-1022. 

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