Inbiochemistry,immunostaining is any use of anantibody-based method to detect a specificprotein in a sample. The term "immunostaining" was originally used to refer to theimmunohistochemical staining of tissue sections, as first described byAlbert Coons in 1941.[1] However, immunostaining now encompasses a broad range of techniques used inhistology,cell biology, andmolecular biology that use antibody-based staining methods.
Immunohistochemistry or IHC staining oftissue sections (orimmunocytochemistry, which is the staining ofcells), is perhaps the most commonly applied immunostaining technique.[2] While the first cases of IHC staining usedfluorescentdyes (seeimmunofluorescence), other non-fluorescent methods usingenzymes such asperoxidase (seeimmunoperoxidase staining) andalkaline phosphatase are now used. These enzymes are capable of catalysing reactions that give a coloured product that is easily detectable by lightmicroscopy. Alternatively,radioactiveelements can be used as labels, and the immunoreaction can be visualized byautoradiography.[3]
Tissue preparation orfixation 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 informalin-fixedparaffin-embedded tissue sections. However, some antigens will not survive even moderate amounts of aldehyde fixation. Under these conditions, tissues should be rapidly fresh frozen inliquid 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 byantigen 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 negativecontrols for staining are essential for determining specificity.[citation needed]
Aflow 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 anypurification steps. Proteins are generally separated by size usinggel electrophoresis before being transferred to asyntheticmembrane 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 usingperoxidase linked antibodies to catalyse achemiluminescent 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 proteinmolecular 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 fromblood 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 usingtransmission 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. Proteinbiotinylationin 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, anantibody is used to detect a specificproteinepitope. These antibodies can bemonoclonal orpolyclonal. Detection of this first orprimary antibody can be accomplished in multiple ways.
As previously described, enzymes such ashorseradishperoxidase oralkaline phosphatase are commonly used to catalyse reactions that give a coloured orchemiluminescent product.Fluorescent molecules can be visualised usingfluorescence microscopy orconfocal microscopy.[citation needed]
The applications of immunostaining are numerous, but are most typically used inclinical diagnostics andlaboratory research.[citation needed]
Clinically, IHC is used inhistopathology 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.