Actin staining techniques in fixed and living cells

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…]

2 new Actin and Tubulin live-cell imaging stains – without transfection!

Cytoskeletal live-cell imaging is extremely powerful when investigating cellular processes such as cytokinesis, motility and organelle transport and organization. The current experimental procedures remain nevertheless cumbersome and long. This post demonstrates how cell permeable, transfection free, Tubulin and Actin red fluorescent dyes help Cell biologists in analysing cytoskeleton dynamics in living cells.

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