Information about Fixation (histology)
In the fields of histology, pathology, and cell biology, fixation is a chemical process by which biological tissues are preserved from decay. Fixation terminates any ongoing biochemical reactions, and may also increase the mechanical strength or stability of the treated tissues.
First, a fixative usually acts to disable intrinsic biomolecules – particularly proteolytic enzymes – which would otherwise digest or damage the sample.
Second, a fixative will typically protect a sample from extrinsic damage. Fixatives are toxic to most common microorganisms (bacteria in particular) which might exist in a tissue sample or which might otherwise colonise the fixed tissue. In addition, many fixatives will chemically alter the fixed material to make it less palatable (either indigestible or toxic) to opportunistic microorganisms.
Finally, fixatives often alter the cells or tissues on a molecular level to increase their mechanical strength or stability. This increased strength and rigidity can help preserve the morphology (shape and structure) of the sample as it is processed for further analysis.
Perfusion: Fixation via bloodflow. The fixative is injected into the heart with the injection volume matching cardiac output. The fixative spreads through the entire body, and the tissue doesn't die until it is fixed. This has the advantage of preserving perfect morphology, but the disadvantages that the subject dies and the cost is high (because of the volume of fixative needed for larger organisms)
Immersion: The sample of tissue is immersed in fixative of volume at a minimum of 2/3rds greater than the volume of the tissue to be fixed. The fixative must diffuse through the tissue in order to fix, so tissue size and density, as well as the type of fixative must be taken into account. Using a larger sample means it will take longer for the fixative to reach the deeper tissue.
Another popular aldehyde for fixation is glutaraldehyde. It is believed to operate by a similar mechanism to formaldehyde. As a somewhat larger molecule, glutaraldehyde may not penetrate thicker tissue specimens as effectively as formaldehyde. On the other hand, glutaraldehyde may offer a more rigid or tightly linked fixed product—its greater length and two aldehyde groups allow it to 'bridge' and link more distant pairs of protein molecules.
Some fixation protocols call for a combination of formaldehyde and glutaraldehyde, so that their respective strengths complement one another.
These crosslinking fixatives – especially formaldehyde – tend to preserve the secondary structure of proteins and may protect significant amounts of tertiary structure as well.
Osmium tetroxide is often used as a secondary fixative when samples are prepared for electron microscopy. (It is not used for light microscopy as it penetrates thick sections of tissue very poorly.)
Potassium dichromate, chromic acid, and potassium permanganate all find use in certain specific histological preparations.
The most common precipitating fixatives are ethanol and methanol. Acetone is also used.
Acetic acid is a denaturant that is sometimes used in combination with the other precipitating fixatives. The alcohols, by themselves, are known to cause shrinkage of tissue during fixation while acetic acid alone is associated with tissue swelling; combining the two may result in better preservation of tissue morphology.
Pathology is the study and diagnosis of disease through examination of organs, tissues, cells and bodily fluids.
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Purpose of fixation
The purpose of fixation is to preserve a sample of biological material (tissue or cells) to permit stable storage and analysis. To achieve this goal, several conditions must usually be met.First, a fixative usually acts to disable intrinsic biomolecules – particularly proteolytic enzymes – which would otherwise digest or damage the sample.
Second, a fixative will typically protect a sample from extrinsic damage. Fixatives are toxic to most common microorganisms (bacteria in particular) which might exist in a tissue sample or which might otherwise colonise the fixed tissue. In addition, many fixatives will chemically alter the fixed material to make it less palatable (either indigestible or toxic) to opportunistic microorganisms.
Finally, fixatives often alter the cells or tissues on a molecular level to increase their mechanical strength or stability. This increased strength and rigidity can help preserve the morphology (shape and structure) of the sample as it is processed for further analysis.
Fixation process
Fixation is usually the first stage in a multistep process to prepare a sample of biological material for microscopy or other analysis. Therefore, the choice of fixative and fixation protocol may depend on the additional processing steps and final analyses that are planned. For example, immunohistochemistry utilises antibodies which bind to a specific protein target. Prolonged fixation can chemically mask these targets and prevent antibody binding. In these cases, a 'quick fix' method using cold formalin for around 24 hours is typically used.Types of fixation
There are generally two types of fixation process:Perfusion: Fixation via bloodflow. The fixative is injected into the heart with the injection volume matching cardiac output. The fixative spreads through the entire body, and the tissue doesn't die until it is fixed. This has the advantage of preserving perfect morphology, but the disadvantages that the subject dies and the cost is high (because of the volume of fixative needed for larger organisms)
Immersion: The sample of tissue is immersed in fixative of volume at a minimum of 2/3rds greater than the volume of the tissue to be fixed. The fixative must diffuse through the tissue in order to fix, so tissue size and density, as well as the type of fixative must be taken into account. Using a larger sample means it will take longer for the fixative to reach the deeper tissue.
Types of fixatives
Crosslinking fixatives
Crosslinking fixatives act by creating covalent chemical bonds between proteins in tissue. This anchors soluble proteins to the cytoskeleton, and lends additional rigidity to the tissue.Aldehydes
By far the most commonly used fixative in histology is the crosslinking fixative formaldehyde (often sold as a saturated aqueous solution under the name formalin). Formaldehyde is thought to interact primarily with the residues of the basic amino acid lysine.Another popular aldehyde for fixation is glutaraldehyde. It is believed to operate by a similar mechanism to formaldehyde. As a somewhat larger molecule, glutaraldehyde may not penetrate thicker tissue specimens as effectively as formaldehyde. On the other hand, glutaraldehyde may offer a more rigid or tightly linked fixed product—its greater length and two aldehyde groups allow it to 'bridge' and link more distant pairs of protein molecules.
Some fixation protocols call for a combination of formaldehyde and glutaraldehyde, so that their respective strengths complement one another.
These crosslinking fixatives – especially formaldehyde – tend to preserve the secondary structure of proteins and may protect significant amounts of tertiary structure as well.
Oxidising agents
The oxidising fixatives can react with various side chains of proteins and other biomolecules, allowing the formation of crosslinks which stabilise tissue structure.Osmium tetroxide is often used as a secondary fixative when samples are prepared for electron microscopy. (It is not used for light microscopy as it penetrates thick sections of tissue very poorly.)
Potassium dichromate, chromic acid, and potassium permanganate all find use in certain specific histological preparations.
Precipitating fixatives
Precipitating (or denaturing) fixatives act by reducing the solubility of protein molecules and (often) by disrupting the hydrophobic interactions which give many proteins their tertiary structure. The precipitation and aggregation of proteins is a very different process from the crosslinking which occurs with the aldehyde fixatives.The most common precipitating fixatives are ethanol and methanol. Acetone is also used.
Acetic acid is a denaturant that is sometimes used in combination with the other precipitating fixatives. The alcohols, by themselves, are known to cause shrinkage of tissue during fixation while acetic acid alone is associated with tissue swelling; combining the two may result in better preservation of tissue morphology.
Other fixatives
Other fixative agents include picric acid and mercuric chloride.See also
Histology (from the Greek ἱστός) is the study of tissue sectioned as a thin slice, using a microtome. It can be described as microscopic anatomy.
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Pathologist redirects here. For other uses of the terms pathology or pathological, see pathology (disambiguation).
Pathology is the study and diagnosis of disease through examination of organs, tissues, cells and bodily fluids.
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Cell biology (also called cellular biology or formerly cytology, from the Greek kytos, "container") is an academic discipline that studies cells. This includes their physiological properties, their structure, the organelles they contain, interactions with
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Biological tissue is a collection of interconnected cells that perform a similar function within an organism.
The study of tissue is known as histology, or, in connection with disease, histopathology.
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The study of tissue is known as histology, or, in connection with disease, histopathology.
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Biological tissue is a collection of interconnected cells that perform a similar function within an organism.
The study of tissue is known as histology, or, in connection with disease, histopathology.
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The study of tissue is known as histology, or, in connection with disease, histopathology.
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Proteolysis is the directed degradation (digestion) of proteins by cellular enzymes called proteases or by intramolecular digestion.
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Purposes
Proteolysis is used by the cell for several purposes...... Click the link for more information.
Enzymes are proteins that catalyze (i.e. accelerate) chemical reactions.[1] In enzymatic reactions, the molecules at the beginning of the process are called substrates, and the enzyme converts them into different molecules, the products.
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Bacteria
Phyla
Actinobacteria
Aquificae
Chlamydiae
Bacteroidetes/Chlorobi
Chloroflexi
Chrysiogenetes
Cyanobacteria
Deferribacteres
Deinococcus-Thermus
Dictyoglomi
Fibrobacteres/Acidobacteria
Firmicutes
Fusobacteria
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Phyla
Actinobacteria
Aquificae
Chlamydiae
Bacteroidetes/Chlorobi
Chloroflexi
Chrysiogenetes
Cyanobacteria
Deferribacteres
Deinococcus-Thermus
Dictyoglomi
Fibrobacteres/Acidobacteria
Firmicutes
Fusobacteria
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The term morphology in biology refers to the outward appearance (shape, structure, color, pattern) of an organism or taxon and its component parts. This is in contrast to physiology, which deals primarily with function.
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Microscopy is any technique for producing visible images of structures or details too small to otherwise be seen by the human eye, using a microscope or other magnification tool. It is often used more specifically as a technique of using a microscope.
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Covalent bonding is a form of chemical bonding that is characterized by the sharing of pairs of electrons between atoms, or between atoms and other covalent bonds.
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cytoskeleton is a cellular "scaffolding" or "skeleton" contained, as all other organelles, within the cytoplasm. It is contained in all eukaryotic cells and recent research has shown it can be present in prokaryotic cells too.
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Formaldehyde (methanal) is the chemical compound with the formula H2CO. The simplest aldehyde, it was first synthesized by the Russian chemist Aleksandr Butlerov but was conclusively identified by August Wilhelm von Hofmann.
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Lysine (abbreviated as Lys or K)[1] is an α-amino acid with the chemical formula HO2CCH(NH2)(CH2)4NH2.
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aldehyde is an organic compound containing a terminal carbonyl group. This functional group, which consists of a carbon atom which is bonded to a hydrogen atom and double-bonded to an oxygen atom (chemical formula O=CH-), is called the aldehyde group.
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Glutaraldehyde is a colorless liquid with a pungent odor used to sterilize medical and dental equipment. It is also used for industrial water treatment and as a chemical preservative.
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secondary structure is the general three-dimensional form of local segments of biopolymers such as proteins and nucleic acids (DNA/RNA). It does not, however, describe specific atomic positions in three-dimensional space, which are considered to be tertiary structure.
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Proteins are large organic compounds made of amino acids arranged in a linear chain and joined together by peptide bonds between the carboxyl and amino groups of adjacent amino acid residues.
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In biochemistry and chemistry, the tertiary structure of a protein or any other macromolecule is its three-dimensional structure, as defined by the atomic coordinates.[1]
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Relationship to primary sequence
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Osmium tetroxide is the chemical compound with the formula OsO4. The compound is noteworthy for its many uses, despite the rarity of osmium. It also has a number of interesting properties, one being that the solid is volatile.
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An electron microscope is a type of microscope that uses electrons as a way to illuminate and create an image of a specimen. It has much higher magnification and resolving power than a light microscope, with magnifications up to about two million times, compared to about two
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Potassium dichromate, K2Cr2O7, is a common inorganic chemical reagent, most commonly used as an oxidising agent in various laboratory and industrial applications.
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Chromic acid refers to a collection of compounds generated by the acidification of solutions containing chromate and dichromate anions or the dissolving of chromium trioxide in sulfuric acid.
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Potassium permanganate is the chemical compound KMnO4. In this salt, manganese is in the +7 oxidation state. The salt is also known as "permanganate of potash." The permanganate ion is a strong oxidizing agent.
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hydrophobicity (from the combining form of water in Attic Greek hydro- and for fear phobos) refers to the physical property of a molecule (known as a hydrophobe) that is repelled from a mass of water [1].
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Precipitation is the formation of a solid in a solution during a chemical reaction. When the chemical reaction occurs the solid formed is called the precipitate. This can occur when an insoluble substance, the precipitate
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Ethanol, also known as ethyl alcohol, drinking alcohol or grain alcohol, is a flammable, colorless, slightly toxic chemical compound, and is best known as the alcohol found in alcoholic beverages.
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Methanol, also known as methyl alcohol, carbinol, wood alcohol, wood naptha or wood spirits, is a chemical compound with chemical formula CH3OH.
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The chemical compound acetone (also known as propanone, dimethyl ketone, 2-propanone, propan-2-one and β-ketopropane) is the simplest representative of the ketones.
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