Septins are a major component of the mammalian cytoskeleton. Septins associate with filamentous actin (F-actin) and microtubules, but the nature and significance of these interactions are not well understood. Fluorescence microscopy of F-actin- and microtubule-associated septins in fixed and living cells has been instrumental in uncovering septin functions in cellular morphogenesis and cytoskeleton-dependent processes (eg, cell division, cell migration). Here, we provide a detailed methodology for the visualization of endogenous septins by immunofluorescence microscopy, discussing sample preparation and reagents that are critical for optimal staining. In addition, we review approaches for the construction and expression of fluorescent septins and their time-lapse imaging with F-actin and microtubules. The recommended methodology is adaptable for high- and superresolution imaging of mammalian cells with various instrumentation, including wide-field and confocal microscopy as well as total internal reflection fluorescence and structured illumination microscopy.
Keywords: Actin; Confocal microscopy; Cytoskeleton; Fluorescence imaging; Immunofluorescence; Live cell imaging; Microtubules; Septins; Superresolution microscopy; Time-lapse microscopy.
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