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. Biochemistry is the study of the chemical processes in living Organisms It deals with the Structure and function of cellular components such as Antibodies (also known as immunoglobulins, abbreviated Ig) are Gamma globulin Proteins that are found in Blood or other Bodily Proteins are large Organic compounds made of Amino acids arranged in a linear chain and joined together by Peptide bonds between the Carboxyl The term immunostaining was originally used to refer to the immunohistochemical staining of tissue sections, as first described by Albert Coons in 1941. Immunohistochemistry or IHC refers to the process of localizing proteins in cells of a tissue section exploiting the principle of antibodies binding specifically ^[1] Now however, immunostaining encompasses a broad range of techniques used in histology, cell biology, and molecular biology that utilise antibody-based staining methods. Histology (from the Greek = 'tissue' is the study of the microscopic anatomy of cells and tissues of Plants and See also List of basic cell biology topics. Cell biology (also called cellular biology or formerly cytology, from the Molecular biology is the study of Biology at a molecular level

Contents 1 Immunostaining Techniques 1.1 Immunohistochemistry 1.2 Flow cytometry 1.3 Western blotting 1.4 Enzyme-linked immunosorbent assay 1.5 Immuno-electron microscopy 2 Methodological overview 3 Applications of Immunostaining 4 References

Immunostaining Techniques

Immunohistochemistry

Main article: Immunohistochemistry

Immunohistochemistry or IHC staining of cells or tissue sections is perhaps the most commonly applied immunostaining technique. Immunohistochemistry or IHC refers to the process of localizing proteins in cells of a tissue section exploiting the principle of antibodies binding specifically The cell is the structural and functional unit of all known living Organisms It is the smallest unit of an organism that is classified as living and is often called Tissue is a cellular organizational level intermediate between cells and a complete organism ^[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. Fluorescence is a Luminescence that is mostly found as an A dye can generally be described as a Colored substance that has an affinity to the substrate to which it is being applied Immunofluorescence is the labeling of antibodies or Antigens with fluorescent Dyes This technique is often used to visualize the subcellular Enzymes are Biomolecules that catalyze ( ie increase the rates of Chemical reactions Almost all enzymes are Proteins Peroxidases ( EC number 1111x are a large family of Enzymes A majority of peroxidase protein sequences can be found in the PeroxiBase database Immunoperoxidase is a type of immunostain used in Molecular biology, medical research and clinical diagnostics Alkaline phosphatase ( ALP) ( is a Hydrolase Enzyme responsible for removing Phosphate groups from many types of molecules including These enzymes are capable of catalysing reactions that give a coloured product that is easily detectable by light microscopy. Microscopy is the technical field of using microscopes to view samples or objects Alternatively, radioactive elements can be used as labels, and the immunoreaction can be visualized by autoradiography. Radioactive decay is the process in which an unstable Atomic nucleus loses energy by emitting ionizing particles and Radiation. A chemical element is a type of Atom that is distinguished by its Atomic number; that is by the number of Protons in its nucleus. An autoradiograph is an image on an X-ray film or nuclear emulsion produced by the pattern of decay emissions (e ^[3]

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. Formaldehyde is a Chemical compound with the formula H2CO It is the simplest Aldehyde —an organic compound containing a terminal Carbonyl In chemistry paraffin is the common name for the Alkane Hydrocarbons with the general formula C n H2 n +2 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. Liquid nitrogen (liquid density at the Triple point is 0707 g/mL is the liquid produced industrially in large quantities by Fractional distillation of 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 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 and negative controls for staining are essential for determining specificity. Scientific controls allow Experiments to study one Variable at a time and are a vital part of the Scientific method.

Flow cytometry

Main article: flow cytometry

A flow cytometer can be used for the direct analysis of cells expressing one or more specific proteins. 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 Cells are immunostained in solution using methods similar to used for immunofluorescence, and then analysed by flow cytometry.

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. The western blot (alternatively immunoblot) is an Analytical technique used to detect specific Proteins in a given sample of tissue homogenate or Protein purification is a series of processes intended to isolate a single type of Protein from a complex mixture Proteins are generally separated by size using gel electrophoresis before being transferred to a synthetic membrane via dry, semi-dry, or wet blotting methods. 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 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. Peroxidases ( EC number 1111x are a large family of Enzymes A majority of peroxidase protein sequences can be found in the PeroxiBase database Chemiluminescence (sometimes " chemoluminescence " is the emission of Light ( Luminescence) with limited emission of heat as the result of a chemical

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 (abbreviated m of a substance, more commonly referred to as molecular weight and abbreviated as MW, is the Mass of one

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). Enzyme-Linked ImmunoSorbent Assay, also called ELISA, Enzyme ImmunoAssay or EIA, is a biochemical technique used mainly in Immunology Blood plasma is the Liquid component of Blood, in which the Blood cells are suspended 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.

Immuno-electron microscopy

Electron microscopy or EM can be used to study the detailed microarchitecture of tissues or cells. An electron microscope is a type of Microscope that uses Electrons to illuminate a specimen and create an enlarged image 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.

Methodological overview

In immunostaining methods, an antibody is used to detect a specific protein epitope. Antibodies (also known as immunoglobulins, abbreviated Ig) are Gamma globulin Proteins that are found in Blood or other Bodily Proteins are large Organic compounds made of Amino acids arranged in a linear chain and joined together by Peptide bonds between the Carboxyl An epitope, also known as antigenic determinant, is the part of a Macromolecule that is recognized by the Immune system, specifically by antibodies These antibodies can be monoclonal or polyclonal. Monoclonal antibodies ( mAb or moAb) are monospecific antibodies that are identical because they are produced by one type of immune cell Polyclonal antibodies (or antisera are antibodies that are derived from different B cell lines Detection of this first or primary antibody can be accomplished in multiple ways.

• The primary antibody can be directly labeled using an enzyme or fluorophore. Enzymes are Biomolecules that catalyze ( ie increase the rates of Chemical reactions Almost all enzymes are Proteins A fluorophore, in analogy to a Chromophore, is a component of a molecule which causes a molecule to be Fluorescent.
• 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. Vitamin H redirects here In medical slang "vitamin H" may also refer to Haloperidol.
• The primary antibody can be probed for using a broader species-specific secondary antibody that is labeled using an enzyme, or fluorophore. A secondary antibody is an Antibody that binds to Primary antibodies or antibody fragments
• In the case of electron microscopy, antibodies are linked to a heavy metal atom. An electron microscope is a type of Microscope that uses Electrons to illuminate a specimen and create an enlarged image History See also Atomic theory, Atomism The concept that matter is composed of discrete units and cannot be divided into arbitrarily tiny

As previously described, enzymes such as horseradish peroxidase or alkaline phosphatase are commonly used to catalyse reactions that give a coloured or chemiluminescent product. Horseradish ( Armoracia rusticana, syn Cochlearia armoracia) is a Perennial plant of the Brassicaceae family which also includes mustard Peroxidases ( EC number 1111x are a large family of Enzymes A majority of peroxidase protein sequences can be found in the PeroxiBase database Alkaline phosphatase ( ALP) ( is a Hydrolase Enzyme responsible for removing Phosphate groups from many types of molecules including Chemiluminescence (sometimes " chemoluminescence " is the emission of Light ( Luminescence) with limited emission of heat as the result of a chemical Fluorescent molecules can be visualised using fluoresence microscopy or confocal microscopy. Fluorescence is a Luminescence that is mostly found as an Confocal microscopy is an optical imaging technique used to increase Micrograph contrast and/or to Reconstruct three-dimensional Images by

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 (from the Greek histos (tissue and pathos (suffering refers to the microscopic examination of tissue in order to study the manifestations

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

© 2009 citizendia.org; parts available under the terms of GNU Free Documentation License, from http://en.wikipedia.org
Dapyx Software network: MP3 Explorer | Ebook Manager | Zenithic