免疫蛍光法（Immunofluorescence, IF）

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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References:

1. 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