![]() ![]() However, concentrates do require preparation time and validation. Given the large working dilution range, concentrates can be varied at any time to accommodate changes to laboratory practice or for multiple protocols for a particular antibody. The working dilution of concentrates can be optimized to balance cost, staining time, and quality. Concentrates are flexible, have a lower initial purchase price, and can generally be used within any staining system, both automated and manual, subject to the manufacturer’s recommendations. When selecting antibodies, there are two main options to consider a concentrated format or a pre-diluted, Ready-To-Use (RTU) format. Exposure to air and light can cause this. Antibodies over time can lose their staining intensity. Each antibody needs to be tested with your staining system. What might work well in one laboratory might not be optimal for your laboratory. There are several things to keep in mind with antibodies. Search for specific antibodies from literature, vendors, as well as your peers. This can save you a lot of headaches down the road. The counter-stain provides a contrast to the chromogen and also helps the pathologist visualize the underlying tissue structure.Ī simple, but sometimes overlooked step is to choose antibodies that work for immunohistochemistry. The blue background is a hematoxylin counter-stain that is often applied after the chromogen. The amount of staining, the staining pattern, and the location of staining (cell cytoplasm nucleus or membrane) all provide information for the diagnosing pathologist. This is a typical IHC stain where the brown precipitate indicates the presence of the target antigen – in this case, Cytokeratin 5 on a prostate biopsy. This is a process known as double staining. Stain precipitate skin#AP Red (or another red chromogen) is used mainly for skin sections where the brown DAB may be masked by brown melanin pigment.īoth DAB and AP Red are sometimes used in the same tissue section to allow the pathologist to visualize two antigens in the one slide. There are two commonly used chromogens: DAB (brown) or AP (red).ĭAB is used for most applications as it provides strong and permanent stains. The Chromogenįinally, a substrate forms an insoluble colored precipitate that can be visualized under a microscope. Multiple enzymes attached to the antibody are known as polymers, and they again produce more intense staining as there are more molecules for the chromogen to attach. Modern chromogenic detection utilizes enzymes such as Horseradish Peroxidase (HRP) that are conjugated (joined) to an antibody. The detection system builds on the secondary. It is now commonly used as multiple secondaries can bind to a single primary to amplify the staining intensity. Next, secondary antibodies bind to the primary antibody. Monoclonal antibodies have an affinity to only one epitope and tend to produce cleaner, more specific staining but are less sensitive or intense. Polyclonal antibodies have an affinity with, and bind to, multiple epitopes (or parts) or the target antigen, and as such are more prone to cross-react to non-target antigens. There are two main types of antibody, polyclonal and monoclonal. The first stage of IHC is the application of a primary antibody that binds specifically to the target antigen. Often, a pathologist will use a “panel” of multiple antigens to help fully classify a particular tumor. There are many hundreds of antigens that have been found to be diagnostically useful. Pathologists look for the presence or absence of particular antigens to assist with diagnosis. Target AntigenĪntigens are proteins that are within or on the surface of a cell. IHC is used as a diagnostic tool to assist in the diagnosis of solid tumors and cytological specimens and has been used as a mainstream diagnostic tool for almost half a century. Where H&E and Special Stains are non-specific, IHC is directed to a specific protein marker or markers. IHC has evolved to complement the Hematoxylin & Eosin (H&E) and Special Stain techniques that typically show tissue morphology (structure). IHC is used in histology to detect the presence of specific protein markers that can assist with accurate tumor classification and diagnosis. ![]()
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