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Acting and reacting in life science biotechnologies

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9 reagents to visualize actin in fixed cells

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2015 was marked by the increasing number of studies related to cellular cytoskeletal dynamics. In this context, it should be noted that the new generation of live cell-imaging SiR-Actin & SiR-Tubulin stains have been widely embraced by Life Scientists. Launched a few months ago, these fluorescent probes are now regularly cited in the “Material & Methods” section of numerous prestigious publications as indicated by Ali (see his the posts “Live cell imaging tool SiR-actin on JBC frontcover” and “User experience of SiR-Actin and SiR-Tubulin Live Cell Imaging“).

Nevertheless, cytoskeletal interactions are still widely studied in “fixed” cells. In the same manner as for live cell-imaging, these experimental assays require high quality reagents too. With the help of our friends from Cystoskeleton Inc., we have selected 9 premium research tools validated for actin visualization in PFA-fixed cells. [Read more…]

Actin staining techniques in fixed and living cells

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Actin can be stained in living and fixed cells to determine and follow the structure and function of the cytoskeleton. The actin cytoskeleton is a very dynamic and labile structure in the living cell, but it can be fixed by either cold methanol or paraformaldehyde prior to probing or staining for actin structures.

Actin staining in fixed cells

Phalloidin - Actin binding

Fluorescent phalloidin binding to F-Actin, Source: Cytoskeleton Inc.

In fixed cells, actin structures can be visualized by actin antibodies, fluorescent phalloidins, or even electron microscopy.
Antibodies recognize both monomer and polymer (filamentous or F-actin) actin and hence tend to have a high background compared to probes that bind only F-actin. Well designed fluorescent phalloidins only bind to the native quaternary structure of F-actin and therefore have a low background. To create the correct fixation conditions for phalloidin binding, paraformaldehyde must be used as the fixative because it retains the quanternary protein structure which is necessary for high affinity. Methanol destroys the native conformation and hence is not suitable for actin staining with phalloidin. [Read more…]