Immunostaining
Encyclopedia
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. Now however, immunostaining encompasses a broad range of techniques used in histology
, cell biology
, and molecular biology
that utilise antibody-based staining methods.
sections (or immunocytochemistry
, which is the staining of cell
s), is perhaps the most commonly applied immunostaining technique. While the first cases of IHC staining used fluorescent
dye
s (see immunofluorescence
), other non-fluorescent methods using enzyme
s 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 autoradiograph
y.
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 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.
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. Flow cytometry also has a high capital cost associated with the purchase of a flow cytometer.
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.
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.
, 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.
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.
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.
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 fluorescence microscopy or confocal microscopy
.
Clinically, IHC is used in histopathology
for the diagnosis of specific types of cancers based on molecular markers.
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.
Biochemistry
Biochemistry, sometimes called biological chemistry, is the study of chemical processes in living organisms, including, but not limited to, living matter. Biochemistry governs all living organisms and living processes...
that applies to any use of an antibody
Antibody
An antibody, also known as an immunoglobulin, is a large Y-shaped protein used by the immune system to identify and neutralize foreign objects such as bacteria and viruses. The antibody recognizes a unique part of the foreign target, termed an antigen...
-based method to detect a specific protein
Protein
Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...
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. Now however, immunostaining encompasses a broad range of techniques used in histology
Histology
Histology is the study of the microscopic anatomy of cells and tissues of plants and animals. It is performed by examining cells and tissues commonly by sectioning and staining; followed by examination under a light microscope or electron microscope...
, cell biology
Cell biology
Cell biology is a scientific discipline that studies cells – their physiological properties, their structure, the organelles they contain, interactions with their environment, their life cycle, division and death. This is done both on a microscopic and molecular level...
, and molecular biology
Molecular biology
Molecular biology is the branch of biology that deals with the molecular basis of biological activity. This field overlaps with other areas of biology and chemistry, particularly genetics and biochemistry...
that utilise antibody-based staining methods.
Immunohistochemistry
Immunohistochemistry or IHC staining of tissueTissue (biology)
Tissue is a cellular organizational level intermediate between cells and a complete organism. A tissue is an ensemble of cells, not necessarily identical, but from the same origin, that together carry out a specific function. These are called tissues because of their identical functioning...
sections (or immunocytochemistry
Immunocytochemistry
Immunocytochemistry is a common laboratory technique that uses antibodies that target specific peptides or protein antigens in the cell via specific epitopes. These bound antibodies can then be detected using several different methods. ICC allows researchers to evaluate whether or not cells in a...
, which is the staining of cell
Cell (biology)
The cell is the basic structural and functional unit of all known living organisms. It is the smallest unit of life that is classified as a living thing, and is often called the building block of life. The Alberts text discusses how the "cellular building blocks" move to shape developing embryos....
s), is perhaps the most commonly applied immunostaining technique. While the first cases of IHC staining used fluorescent
Fluorescence
Fluorescence is the emission of light by a substance that has absorbed light or other electromagnetic radiation of a different wavelength. It is a form of luminescence. In most cases, emitted light has a longer wavelength, and therefore lower energy, than the absorbed radiation...
dye
Dye
A dye is a colored substance that has an affinity to the substrate to which it is being applied. The dye is generally applied in an aqueous solution, and requires a mordant to improve the fastness of the dye on the fiber....
s (see immunofluorescence
Immunofluorescence
Immunofluorescence is a technique used for light microscopy with a fluorescence microscope and is used primarily on biological samples. This technique uses the specificity of antibodies to their antigen to target fluorescent dyes to specific biomolecule targets within a cell, and therefore allows...
), other non-fluorescent methods using enzyme
Enzyme
Enzymes are proteins that catalyze chemical reactions. In enzymatic reactions, the molecules at the beginning of the process, called substrates, are converted into different molecules, called products. Almost all chemical reactions in a biological cell need enzymes in order to occur at rates...
s such as peroxidase
Peroxidase
Peroxidases are a large family of enzymes that typically catalyze a reaction of the form:For many of these enzymes the optimal substrate is hydrogen peroxide, but others are more active with organic hydroperoxides such as lipid peroxides...
(see immunoperoxidase staining
Immunoperoxidase
Immunoperoxidase is a type of immunostain used in molecular biology, medical research, and clinical diagnostics. In particular, immunoperoxidase reactions refer to a sub-class of immunohistochemical or immunocytochemical procedures in which the antibodies are visualized via a peroxidase-catalyzed...
) and alkaline phosphatase
Alkaline phosphatase
Alkaline phosphatase is a hydrolase enzyme responsible for removing phosphate groups from many types of molecules, including nucleotides, proteins, and alkaloids. The process of removing the phosphate group is called dephosphorylation...
are now used. These enzymes are capable of catalysing reactions that give a coloured product that is easily detectable by light microscopy
Microscopy
Microscopy is the technical field of using microscopes to view samples and objects that cannot be seen with the unaided eye...
. Alternatively, radioactive
Radioactive decay
Radioactive decay is the process by which an atomic nucleus of an unstable atom loses energy by emitting ionizing particles . The emission is spontaneous, in that the atom decays without any physical interaction with another particle from outside the atom...
elements
Chemical element
A chemical element is a pure chemical substance consisting of one type of atom distinguished by its atomic number, which is the number of protons in its nucleus. Familiar examples of elements include carbon, oxygen, aluminum, iron, copper, gold, mercury, and lead.As of November 2011, 118 elements...
can be used as labels, and the immunoreaction can be visualized by autoradiograph
Autoradiograph
An autoradiograph is an image on an x-ray film or nuclear emulsion produced by the pattern of decay emissions from a distribution of a radioactive substance...
y.
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
Formaldehyde
Formaldehyde is an organic compound with the formula CH2O. It is the simplest aldehyde, hence its systematic name methanal.Formaldehyde is a colorless gas with a characteristic pungent odor. It is an important precursor to many other chemical compounds, especially for polymers...
-fixed paraffin
Paraffin
In chemistry, paraffin is a term that can be used synonymously with "alkane", indicating hydrocarbons with the general formula CnH2n+2. Paraffin wax refers to a mixture of alkanes that falls within the 20 ≤ n ≤ 40 range; they are found in the solid state at room temperature and begin to enter the...
-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
Liquid nitrogen
Liquid nitrogen is nitrogen in a liquid state at a very low temperature. It is produced industrially by fractional distillation of liquid air. Liquid nitrogen is a colourless clear liquid with density of 0.807 g/mL at its boiling point and a dielectric constant of 1.4...
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
Vibratome
A vibratome is an instrument that is similar to a microtome but uses a vibrating razor blade to cut through tissue. The vibration amplitude, the speed, and the angle of the blade can all be controlled...
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
Scientific control
Scientific control allows for comparisons of concepts. It is a part of the scientific method. Scientific control is often used in discussion of natural experiments. For instance, during drug testing, scientists will try to control two groups to keep them as identical and normal as possible, then...
for staining are essential for determining specificity.
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 those 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. Flow cytometry also has a high capital cost associated with the purchase of a flow cytometer.
Western blotting
Western blotting allows the detection of specific proteins from extracts made from cells or tissues, before or after any purificationProtein purification
Protein purification is a series of processes intended to isolate a single type of protein from a complex mixture. Protein purification is vital for the characterization of the function, structure and interactions of the protein of interest. The starting material is usually a biological tissue or...
steps. Proteins are generally separated by size using gel electrophoresis
Gel electrophoresis
Gel electrophoresis is a method used in clinical chemistry to separate proteins by charge and or size and in biochemistry and molecular biology to separate a mixed population of DNA and RNA fragments by length, to estimate the size of DNA and RNA fragments or to separate proteins by charge...
before being transferred to a synthetic
Chemical synthesis
In chemistry, chemical synthesis is purposeful execution of chemical reactions to get a product, or several products. This happens by physical and chemical manipulations usually involving one or more reactions...
membrane
Artificial membrane
An artificial membrane, or synthetic membrane, is a synthetically created membrane which is usually intended for separation purposes in laboratory or in industry. Synthetic membranes have been successfully used for small and large-scale industrial processes since the middle of twentieth century. A...
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
Peroxidase
Peroxidases are a large family of enzymes that typically catalyze a reaction of the form:For many of these enzymes the optimal substrate is hydrogen peroxide, but others are more active with organic hydroperoxides such as lipid peroxides...
linked antibodies to catalyse a chemiluminescent reaction.
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
Molecular mass
The molecular mass of a substance is the mass of one molecule of that substance, in unified atomic mass unit u...
to be gauged as compared with known molecular weight markers.
Enzyme-linked immunosorbent assay
The enzyme-linked immunosorbent assay or ELISA is a diagnostic method for quantitatively or semi-quantitatively determining protein concentrations from blood plasmaBlood plasma
Blood plasma is the straw-colored liquid component of blood in which the blood cells in whole blood are normally suspended. It makes up about 55% of the total blood volume. It is the intravascular fluid part of extracellular fluid...
, serum
Blood serum
In blood, the serum is the component that is neither a blood cell nor a clotting factor; it is the blood plasma with the fibrinogens removed...
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.
Immuno-electron microscopy
Electron microscopyElectron microscope
An electron microscope is a type of microscope that uses a beam of electrons to illuminate the specimen and produce a magnified image. Electron microscopes have a greater resolving power than a light-powered optical microscope, because electrons have wavelengths about 100,000 times shorter than...
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
Transmission electron microscopy
Transmission electron microscopy is a microscopy technique whereby a beam of electrons is transmitted through an ultra thin specimen, interacting with the specimen as it passes through...
. 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 antibodyAntibody
An antibody, also known as an immunoglobulin, is a large Y-shaped protein used by the immune system to identify and neutralize foreign objects such as bacteria and viruses. The antibody recognizes a unique part of the foreign target, termed an antigen...
is used to detect a specific protein
Protein
Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...
epitope
Epitope
An epitope, also known as antigenic determinant, is the part of an antigen that is recognized by the immune system, specifically by antibodies, B cells, or T cells. The part of an antibody that recognizes the epitope is called a paratope...
. These antibodies can be monoclonal
Monoclonal antibodies
Monoclonal antibodies are monospecific antibodies that are the same because they are made by identical immune cells that are all clones of a unique parent cell....
or polyclonal. Detection of this first or primary antibody can be accomplished in multiple ways.
- The primary antibody can be directly labeled using an enzymeEnzymeEnzymes are proteins that catalyze chemical reactions. In enzymatic reactions, the molecules at the beginning of the process, called substrates, are converted into different molecules, called products. Almost all chemical reactions in a biological cell need enzymes in order to occur at rates...
or fluorophoreFluorophoreA fluorophore, in analogy to a chromophore, is a component of a molecule which causes a molecule to be fluorescent. It is a functional group in a molecule which will absorb energy of a specific wavelength and re-emit energy at a different wavelength...
. - 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 biotinBiotinBiotin, also known as Vitamin H or Coenzyme R, is a water-soluble B-complex vitamin discovered by Bateman in 1916. It is composed of a ureido ring fused with a tetrahydrothiophene ring. A valeric acid substituent is attached to one of the carbon atoms of the tetrahydrothiophene ring...
-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 microscopyElectron microscopeAn electron microscope is a type of microscope that uses a beam of electrons to illuminate the specimen and produce a magnified image. Electron microscopes have a greater resolving power than a light-powered optical microscope, because electrons have wavelengths about 100,000 times shorter than...
, antibodies are linked to a heavy metal atomAtomThe atom is a basic unit of matter that consists of a dense central nucleus surrounded by a cloud of negatively charged electrons. The atomic nucleus contains a mix of positively charged protons and electrically neutral neutrons...
.
As previously described, enzymes such as horseradish
Horseradish
Horseradish is a perennial plant of the Brassicaceae family, which also includes mustard, wasabi, broccoli, and cabbages. The plant is probably native to south eastern Europe and the Arab World , but is popular around the world today...
peroxidase
Peroxidase
Peroxidases are a large family of enzymes that typically catalyze a reaction of the form:For many of these enzymes the optimal substrate is hydrogen peroxide, but others are more active with organic hydroperoxides such as lipid peroxides...
or alkaline phosphatase
Alkaline phosphatase
Alkaline phosphatase is a hydrolase enzyme responsible for removing phosphate groups from many types of molecules, including nucleotides, proteins, and alkaloids. The process of removing the phosphate group is called dephosphorylation...
are commonly used to catalyse reactions that give a coloured or chemiluminescent product. Fluorescent
Fluorescence
Fluorescence is the emission of light by a substance that has absorbed light or other electromagnetic radiation of a different wavelength. It is a form of luminescence. In most cases, emitted light has a longer wavelength, and therefore lower energy, than the absorbed radiation...
molecules can be visualised using fluorescence microscopy or confocal microscopy
Confocal microscopy
Confocal microscopy is an optical imaging technique used to increase optical resolution and contrast of a micrograph by using point illumination and a spatial pinhole to eliminate out-of-focus light in specimens that are thicker than the focal plane. It enables the reconstruction of...
.
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
Histopathology
Histopathology refers to the microscopic examination of tissue in order to study the manifestations of disease...
for the diagnosis of specific types of cancers based on molecular markers.
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.