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Immunohistochemical staining is used to identify specific constituents in tissue sections or immobilized cells. To detect the reaction site, the antibody/antigen complex is labeled with an enzyme that can be reacted with a suitable substrate to give a colored product. Proper fixation is crucial to successful staining. Formaldehyde fixation is often used as the routine initial method of choice for tissue and cell fixation. The antigen (usually immobilized cells or tissue) is fixed and adhered to a glass microscope slide. A primary antibody reacts with the immobilized antigen to form an antigen-antibody complex. A second, biotinylated antibody specific for primary antibody reacts with the complex. Streptavidin conjugated to Peroxidase reacts with the ab-ab-ag complex immobilizing the peroxidase at the site of the antigen. Finally, substrate is added causing a colored precipitate to form on the slide at the location of the antigen. This slide is analyzed using a light microscope or embedded and prepared for electron microscopy.
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