Immunofluorescence (IF) is an immunochemical staining technique that utilizes fluorochrome-labeled antibodies for the visualization and localization of targets, by labelling with a fluorescent dye such as fluorescein isothiocyanate (FITC). It can identify a wide range of components on tissue sections, cultured cells, or individual cells, and is thus highly valuable for their broad capability with the use of combinations of specific antibodies tagged with fluorophores.
There are several steps for IF, with the first being fixation to immobilize target antigens while maintaining cellular architecture. Next, sample preparation and blocking needs to be carried out to prevent non-specific binding of antibodies. Then, incubation of primary and secondary antibodies take place before the final preservation and imaging.
There are two different IF methods available: Direct (Primary) and Indirect (Secondary). Direct detection requires using a single fluorochrome-conjugated primary antibody. Meanwhile, indirect detection uses two antibodies, with the first being an unlabeled primary antibody, followed by a fluorochrome-conjugated secondary antibody.
Although both monoclonal and polyclonal antibodies can be used for IF, they have their own pros and cons. Indirect IF more commonly used as the secondary antibodies are typically polyclonal, allowing for the recognition of multiple epitopes of the targets. However, monoclonal antibodies which are specifically designed for IF may be more preferable if your research involves recognizing very specific targets.1
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Im, K., Mareninov, S., Diaz, M. F. P., & Yong, W. H. (2019). An introduction to Performing Immunofluorescence Staining. Methods in Molecular Biology (Clifton, N.J.), 1897, 299. https://doi.org/10.1007/978-1-4939-8935-5_26
Antibody specificity refers to an antibody's ability to selectively bind to a unique epitope on a target antigen while avoiding interactions with unrelated antigens. This property arises from the highly specialized antigen-binding site located in the variable region of the antibody, which determines its unique binding characteristics.
Antibody affinity refers to the strength of the binding interaction between a single antigen epitope and the paratope (binding site) of an antibody. This interaction is a fundamental measure of how well an antibody recognizes its specific antigen target.
Recombinant antibodies are produced using genetic engineering techniques, unlike traditional antibody production, where the immune system generates antibodies without direct control over their sequence. By introducing genes encoding antibody fragments into host cells, such as bacteria or mammalian cells, recombinant antibodies can be expressed, purified, and deployed for applications including research, diagnostics, and therapeutics.
Recombinant antibody expression is a biotechnological process that involves engineering and producing antibodies outside their natural context using recombinant DNA technology.
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