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. [1] However, immunostaining now encompasses a broad range of techniques used in histology, cell biology, and molecular biology that use antibody-based staining methods.
Immunohistochemistry or IHC staining of tissue sections (or immunocytochemistry, which is the staining of cells), 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]
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.[ citation needed ]
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). [4]
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 both positive and negative controls for staining are essential for determining specificity.[ citation needed ]
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.[ citation needed ]
Flow cytometry has several advantages over IHC including: the ability to define distinct cell populations 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. [5] Flow cytometry also has a high capital cost associated with the purchase of a flow cytometer.[ citation needed ]
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.[ citation needed ]
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.[ citation needed ]
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 absorbed 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.[ citation needed ]
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. Protein biotinylation in vivo was proposed to alleviate the problems caused by frequent incompatibility of antibody staining with fixation protocols that better preserve cell morphology. [6]
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.
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 .[ citation needed ]
The applications of immunostaining are numerous, but are most typically used in clinical diagnostics and laboratory research.[ citation needed ]
Clinically, IHC is used in histopathology for the diagnosis of specific types of cancers based on molecular markers.[ citation needed ]
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 of changes in protein expression or degradation.
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 reaction.
The western blot, 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.
Flow cytometry (FC) is a technique used to detect and measure physical and chemical characteristics of a population of cells or particles.
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 visualization of the distribution of the target molecule through the sample. The specific region an antibody recognizes on an antigen is called an epitope. There have been efforts in epitope mapping since many antibodies can bind the same epitope and levels of binding between antibodies that recognize the same epitope can vary. Additionally, the binding of the fluorophore to the antibody itself cannot interfere with the immunological specificity of the antibody or the binding capacity of its antigen. Immunofluorescence is a widely used example of immunostaining and is a specific example of immunohistochemistry. This technique primarily makes use of fluorophores to visualise the location of the antibodies.
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. Albert Coons conceptualized and first implemented the procedure in 1941.
Hybridoma technology is a method for producing large numbers of identical antibodies. This process starts by injecting a mouse with an antigen that provokes an immune response. A type of white blood cell, the B cell, produces antibodies that bind to the injected antigen. These antibody producing B-cells are then harvested from the mouse and, in turn, fused with immortal B cell cancer cells, a myeloma, to produce a hybrid cell line called a hybridoma, which has both the antibody-producing ability of the B-cell and the longevity and reproductivity of the myeloma. The hybridomas can be grown in culture, each culture starting with one viable hybridoma cell, producing cultures each of which consists of genetically identical hybridomas which produce one antibody per culture (monoclonal) rather than mixtures of different antibodies (polyclonal). The myeloma cell line that is used in this process is selected for its ability to grow in tissue culture and for an absence of antibody synthesis. In contrast to polyclonal antibodies, which are mixtures of many different antibody molecules, the monoclonal antibodies produced by each hybridoma line are all chemically identical.
A fluorescence microscope is an optical microscope that uses fluorescence instead of, or in addition to, scattering, reflection, and attenuation or absorption, to study the properties of organic or inorganic substances. "Fluorescence microscope" refers to any microscope that uses fluorescence to generate an image, whether it is a simple set up like an epifluorescence microscope or a more complicated design such as a confocal microscope, which uses optical sectioning to get better resolution of the fluorescence image.
A direct fluorescent antibody, also known as "direct immunofluorescence", is an antibody that has been tagged in a direct fluorescent antibody test. Its name derives from the fact that it directly tests the presence of an antigen with the tagged antibody, unlike western blotting, which uses an indirect method of detection, where the primary antibody binds the target antigen, with a secondary antibody directed against the primary, and a tag attached to the secondary antibody.
Texas Red or sulforhodamine 101 acid chloride is a red fluorescent dye, used in histology for staining cell specimens, for sorting cells with fluorescent-activated cell sorting machines, in fluorescence microscopy applications, and in immunohistochemistry. Texas Red fluoresces at about 615 nm, and the peak of its absorption spectrum is at 589 nm. The powder is dark purple. Solutions can be excited by a dye laser tuned to 595-605 nm, or less efficiently a krypton laser at 567 nm. The absorption extinction coefficient at 596 nm is about 85,000 M−1cm−1.
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.
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 as proteins and nucleic acids.
Immunolabeling is a biochemical process that enables the detection and localization of an antigen to a particular site within a cell, tissue, or organ. Antigens are organic molecules, usually proteins, capable of binding to an antibody. These antigens can be visualized using a combination of antigen-specific antibody as well as a means of detection, called a tag, that is covalently linked to the antibody. If the immunolabeling process is meant to reveal information about a cell or its substructures, the process is called immunocytochemistry. Immunolabeling of larger structures is called immunohistochemistry.
A reverse phase protein lysate microarray (RPMA) is a protein microarray designed as a dot-blot platform that allows measurement of protein expression levels in a large number of biological samples simultaneously in a quantitative manner when high-quality antibodies are available.
A vibratome is an instrument used to cut thin slices of material. It is similar to a microtome but uses a vibrating blade to cut through tissue. The vibration amplitude, the speed, and the angle of the blade can all be controlled. Fixed or fresh tissue pieces are embedded in low gelling temperature agarose. The resulting agarose block containing the tissue piece is then attached to a metal block and sectioned while submerged in a water or buffer bath. Individual sections are then collected with a fine brush and transferred to slides or multiwell plates for staining.
Fluorescence is used in the life sciences generally as a non-destructive way of tracking or analysing biological molecules. Some proteins or small molecules in cells are naturally fluorescent, which is called intrinsic fluorescence or autofluorescence. Alternatively, specific or general proteins, nucleic acids, lipids or small molecules can be "labelled" with an extrinsic fluorophore, a fluorescent dye which can be a small molecule, protein or quantum dot. Several techniques exist to exploit additional properties of fluorophores, such as fluorescence resonance energy transfer, where the energy is passed non-radiatively to a particular neighbouring dye, allowing proximity or protein activation to be detected; another is the change in properties, such as intensity, of certain dyes depending on their environment allowing their use in structural studies.
Tissues that have been preserved with formaldehyde, a highly reactive compound, contain a variety of chemical modifications that can reduce the detectability of proteins in biomedical procedures such as immunohistochemistry. Antigen retrieval is an approach to reducing or eliminating these chemical modifications. The two primary methods of antigen retrieval are heat-mediated epitope retrieval (HIER) and proteolytic induced epitope retrieval (PIER).
Virus quantification is counting or calculating the number of virus particles (virions) in a sample to determine the virus concentration. It is used in both research and development (R&D) in academic and commercial laboratories as well as in production situations where the quantity of virus at various steps is an important variable that must be monitored. For example, the production of virus-based vaccines, recombinant proteins using viral vectors, and viral antigens all require virus quantification to continually monitor and/or modify the process in order to optimize product quality and production yields and to respond to ever changing demands and applications. Other examples of specific instances where viruses need to be quantified include clone screening, multiplicity of infection (MOI) optimization, and adaptation of methods to cell culture.
Primary and secondary antibodies are two groups of antibodies that are classified based on whether they bind to antigens or proteins directly or target another (primary) antibody that, in turn, is bound to an antigen or protein.
Proximity ligation assay is a technology that extends the capabilities of traditional immunoassays to include direct detection of proteins, protein interactions, extracellular vesicles and post translational modifications with high specificity and sensitivity. Protein targets can be readily detected and localized with single molecule resolution and objectively quantified in unmodified cells and tissues. Utilizing only a few cells, sub-cellular events, even transient or weak interactions, are revealed in situ and sub-populations of cells can be differentiated. Within hours, results from conventional co-immunoprecipitation and co-localization techniques can be confirmed.
A neuronal lineage marker is an endogenous tag that is expressed in different cells along neurogenesis and differentiated cells such as neurons. It allows detection and identification of cells by using different techniques. A neuronal lineage marker can be either DNA, mRNA or RNA expressed in a cell of interest. It can also be a protein tag, as a partial protein, a protein or an epitope that discriminates between different cell types or different states of a common cell. An ideal marker is specific to a given cell type in normal conditions and/or during injury. Cell markers are very valuable tools for examining the function of cells in normal conditions as well as during disease. The discovery of various proteins specific to certain cells led to the production of cell-type-specific antibodies that have been used to identify cells.