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Histochemistry
Immunohistochemistry (IHC) is the most common application of immunostaining. It involves the process of selectively identifying antigens (proteins) in cells of a tissue section by exploiting the principle of antibodies binding specifically to antigens in biological tissues. IHC takes its name from the roots "immuno", in reference to antibodies used in the procedure, and "histo", meaning tissue (compare to immunocytochemistry). Albert Coons conceptualized and first implemented the procedure in 1941. Visualising an antibody-antigen interaction can be accomplished in a number of ways, mainly either of the following: * ''Chromogenic immunohistochemistry'' (CIH), wherein an antibody is conjugated to an enzyme, such as peroxidase (the combination being termed immunoperoxidase), that can catalyse a colour-producing reaction. * '' Immunofluorescence'', where the antibody is tagged to a fluorophore, such as fluorescein or rhodamine. Immunohistochemical staining is widely used in the diag ...
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Fixation (histology)
In the fields of histology, pathology, and cell biology, fixation is the preservation of biological tissues from decay due to autolysis or putrefaction. It terminates any ongoing biochemical reactions and may also increase the treated tissues' mechanical strength or stability. Tissue fixation is a critical step in the preparation of histological sections, its broad objective being to preserve cells and tissue components and to do this in such a way as to allow for the preparation of thin, stained sections. This allows the investigation of the tissues' structure, which is determined by the shapes and sizes of such macromolecules (in and around cells) as proteins and nucleic acids. Purposes In performing their protective role, fixatives denature proteins by coagulation, by forming additive compounds, or by a combination of coagulation and additive processes. A compound that adds chemically to macromolecules stabilizes structure most effectively if it is able to combine with part ...
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Immunostaining
In biochemistry, immunostaining is 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. However, immunostaining now encompasses a broad range of techniques used in histology, cell biology, and molecular biology that use antibody-based staining methods. Techniques Immunohistochemistry Immunohistochemistry or IHC staining of tissue sections (or immunocytochemistry, which is the staining of cells), is perhaps the most commonly applied immunostaining technique. 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 m ...
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Albert Coons
Albert Hewett Coons (June 28, 1912 – September 30, 1978) was an American physician, pathologist, and immunologist. He was the first person to conceptualize and develop immunofluorescent techniques for labeling antibodies in the early 1940s. Early years Coons was born in Gloversville, New York, on June 28, 1912, the son of Albert Selmser and Marion (née Hewett) Coons. His father was the president of a glove-manufacturing company, and his grandfather, Eugene Coons, was a physician. He was educated in Gloversville public schools, graduated with a B.S. from Williams College (Williamstown, Massachusetts) in 1933, and received his M.D. degree from Harvard Medical School in 1937.McDevitt, H. OAlbert Hewett Coons ''Biographical memoirs of the National Academy of Sciences. Thereafter, Albert pursued residency training in internal medicine at Massachusetts General Hospital in Boston, Massachusetts. During the final years of his house-officership, Coons joined the Thorndike Memo ...
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Main Staining Patterns On Immunohistochemistry
Main may refer to: Geography *Main River (other) **Most commonly the Main (river) in Germany *Main, Iran, a village in Fars Province *"Spanish Main", the Caribbean coasts of mainland Spanish territories in the 16th and 17th centuries *''The Main'', the diverse core running through Montreal, Quebec, Canada, also separating the Two Solitudes *Main (lunar crater), located near the north pole of the Moon *Main (Martian crater) People and organisations *Main (surname), a list of people with this family name *Ma'in, alternate spelling for the Minaeans, an ancient people of modern-day Yemen *Main (band), a British ambient band formed in 1991 *Chas. T. Main, an American engineering and hydroelectric company founded in 1893 *MAIN (Mountain Area Information Network), former operator of WPVM-LP (MAIN-FM) in Asheville, North Carolina, U.S. Ships *Main (ship), ''Main'' (ship), an iron sailing ship launched in 1884 *SS Main, SS ''Main'', list of steamships with this name *German sh ...
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Polyclonal Antibodies
Polyclonal antibodies (pAbs) are antibodies that are secreted by different B cell lineages within the body (whereas monoclonal antibodies come from a single cell lineage). They are a collection of immunoglobulin molecules that react against a specific antigen, each identifying a different epitope. Production The general procedure to produce polyclonal antibodies is as follows: # Antigen preparation # Adjuvant selection and preparation # Animal selection # Injection process # Blood serum extraction An antigen/adjuvant conjugate is injected into an animal of choice to initiate an amplified immune response. After a series of injections over a specific length of time, the animal is expected to have created antibodies against the conjugate. Blood is then extracted from the animal and then purified to obtain the antibody of interest. Inoculation is performed on a suitable mammal, such as a mouse, rabbit or goat. Larger mammals are often preferred as the amount of serum that can ...
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Bovine Serum Albumin
Bovine serum albumin (BSA or "Fraction V") is a serum albumin protein derived from cows. It is often used as a protein concentration standard in lab experiments. The nickname "Fraction V" refers to albumin being the fifth fraction of the original Edwin Cohn purification methodology that made use of differential solubility characteristics of plasma proteins. By manipulating solvent concentrations, pH, salt levels, and temperature, Cohn was able to pull out successive "fractions" of blood plasma. The process was first commercialized with human albumin for medical use and later adopted for production of BSA. Properties The full-length BSA precursor polypeptide is 607 amino acids (AAs) in length. An N-terminal 18-residue signal peptide is cut off from the precursor protein upon secretion, hence the initial protein product contains 589 amino acid residues. An additional six amino acids are cleaved to yield the mature BSA protein that contains 583 amino acids. BSA has thre ...
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Western Blot
The western blot (sometimes called the protein immunoblot), or western blotting, is a widely used analytical technique in molecular biology and immunogenetics to detect specific proteins in a sample of tissue homogenate or extract. Besides detecting the proteins, this technique is also utilized to visualize, distinguish, and quantify the different proteins in a complicated protein combination. Western blot technique uses three elements to achieve its task of separating a specific protein from a complex: separation by size, transfer of protein to a solid support, and marking target protein using a primary and secondary antibody to visualize. A synthetic or animal-derived antibody (known as the primary antibody) is created that recognizes and binds to a specific target protein. The electrophoresis membrane is washed in a solution containing the primary antibody, before excess antibody is washed off. A secondary antibody is added which recognizes and binds to the primary antibody ...
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Immunocytochemistry
Immunocytochemistry (ICC) is a common laboratory technique that is used to anatomically visualize the localization of a specific protein or antigen in cells by use of a specific primary antibody that binds to it. The primary antibody allows visualization of the protein under a fluorescence microscope when it is bound by a secondary antibody that has a conjugated fluorophore. ICC allows researchers to evaluate whether or not cells in a particular sample express the antigen in question. In cases where an immunopositive signal is found, ICC also allows researchers to determine which sub-cellular compartments are expressing the antigen. Immuno''cyto''chemistry vs. immuno''histo''chemistry Immunocytochemistry differs from immunohistochemistry in that the former is performed on samples of intact cells that have had most, if not all, of their surrounding extracellular matrix removed. This includes individual cells that have been isolated from a block of solid tissue, cells grown with ...
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Enzymes
Enzymes () are proteins that act as biological catalysts by accelerating chemical reactions. The molecules upon which enzymes may act are called substrate (chemistry), substrates, and the enzyme converts the substrates into different molecules known as product (chemistry), products. Almost all metabolism, metabolic processes in the cell (biology), cell need enzyme catalysis in order to occur at rates fast enough to sustain life. Metabolic pathways depend upon enzymes to catalyze individual steps. The study of enzymes is called ''enzymology'' and the field of pseudoenzyme, pseudoenzyme analysis recognizes that during evolution, some enzymes have lost the ability to carry out biological catalysis, which is often reflected in their amino acid sequences and unusual 'pseudocatalytic' properties. Enzymes are known to catalyze more than 5,000 biochemical reaction types. Other biocatalysts are Ribozyme, catalytic RNA molecules, called ribozymes. Enzymes' Chemical specificity, specific ...
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Biotin
Biotin (or vitamin B7) is one of the B vitamins. It is involved in a wide range of metabolic processes, both in humans and in other organisms, primarily related to the utilization of fats, carbohydrates, and amino acids. The name ''biotin'', borrowed from the German , derives from the Ancient Greek word (; 'life') and the suffix "-in" (a suffix used in chemistry usually to indicate 'forming'). Chemical description Biotin is classified as a heterocyclic compound, with a sulfur-containing ring fused ureido and tetrahydrothiophene group. A C5-carboxylic acid side chain is appended to one of the rings. The ureido ring, containing the −N−CO−N− group, serves as the carbon dioxide carrier in carboxylation reactions. Biotin is a coenzyme for five carboxylase enzymes, which are involved in the catabolism of amino acids and fatty acids, synthesis of fatty acids, and gluconeogenesis. Biotinylation of histone proteins in nuclear chromatin plays a role in chromatin stability and g ...
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Protease
A protease (also called a peptidase, proteinase, or proteolytic enzyme) is an enzyme that catalyzes