Immunofluorescence (in short, IF) is a method in biology that relies on the use of antibodies chemically labeled with fluorescent dyes to visualize molecules under a light microscope. For a successful IF staining, it is crucial to have a good antibody that will specifically detect the antigen(s) within the molecule of interest.
This antibody (often called primary antibody) is covalently linked to a fluorescence dye or will be subsequently bound by a specific second antibody of a different origin (called secondary antibody), which will be labeled with the desired fluorescent dye.
Image credit - dSTORM image of rabbit psoas myofibrils labeled with the titin antibody T12-AF647 (blue). The antibody localizes in a pair of lines, one to on either side of the Z-disk, separated by 160-200nm. Sample provided by Mathias Gautel, Pauline Bennett, and Susan Cox (scale bar: 1µm)
Immunofluorescence is commonly used in molecular and cell biology labs as a robust and simple method to reliably localize molecules on a wide range of fixed cells or tissues. IF staining offers the unique possibility of revealing molecules in their "native" state, minimizing potential perturbations of protein conformation, localization and/or function, which can occur when using fluorescence protein tagging.
Similarly, immunofluorescence is highly informative when studying steady state or endogenous protein levels and localization - fluorescence proteins, on the other hand, can sometimes multimerize or affect the kinetics and expression levels of molecules.
Image credit - dSTORM image of nuclear pores (labelled NuP98-AF647, red) in a U2OS cell, sample prepared by S. Tyagi and U. Manor, Salk Institute, USA (scale bar: 5µm)
The Nanoimager significantly enhances the resolution of fluorescence microscopy to up to 20nm with different super-resolution techniques, including dSTORM, PALM, SIM/confocal, single particle tracking and smFRET. Samples stained using immunofluorescence methods, can be imaged in a fast and automated way using our integrated system, which includes the dedicated NimOS software for image analysis.
The Nanoimager can capture two fluorophores simultaneously (with four different colors) and offers three different illumination modes, epifluorescence, HILO and TIRF, improving signal to noise ratio depending on the thickness of the sample. It also provides unrivalled stability, and thanks to its compact design, it can be used in any lab environment without needing an optical table or designated dark room.
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