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Immunofluorescence (IF) is the labeling of substrates, such as proteins, glycans, or molecules, with fluorescent antibodies. Immunofluorescence can be carried out on tissue sections, single cells, cultures, or suspensions. The sample is usually fixed (killed) and permeabilized to allow antibodies to enter the cell, blocked with a solution that minimizes incorrect binding of the antibody, and then the substrates of interest are bound by an antibody specific to the substrate (primary antibody). If the substrate of interest is on the cell surface IF can be carried out on live cells. IF can be direct, where the substrate is bound by a fluorescently-labeled primary antibody, or indirect, where the unlabeled primary antibody is bound by a second fluorescently-labeled antibody (secondary antibody). This technique is used in fluorescent microscopy to visualize cell components, to image substrates in a gel, for microarrays, and for clinical diagnostic tests. (Credit: Brooke Anderson-White)
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