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. 

Cytoskeleton Inc. offers a set of phalloidin based stains (Acti-Stains) coupled to a number of different fluorophores compatible with popular filter sets such as FITC, TRITC and Cy5. The stains are exceptionally bright and stable and are indeed offered at very economical prices compared to other phalloidin based stains coupled to fluorophores of similar stability.

three colors

Left: Swiss 3T3 cell stained with anti-vinculin (red), Dapi (blue nucleus) and F-actin is stained with Acti-stain™ 488; Mid: Swiss 3T3 cell with activated Rho, nucleus is stained with Dapi (blue) and F-actin is stained with Acti-stain™ 555; Right: Mitotic Swiss 3T3 cell, F-actin stained with Acti-stain™ 670, nuclear DNA stained with Dapi; Source: Cytoskeleton Inc.

There are four colours to choose among:

Currently, there’s a promotion (buy one, get one half price) for these products, offering an interesting opportunity to try them.

Actin staining in living cells

Up to now, the options for live cell staining of Actin (as well as Tubulin) were limited. The only methods for live cell imaging available so far have been systems based on expression vectors which have to be transfected to target cells. In the case of Actin, a binding protein is coupled to either GFP or RFP and thus is expressed in target cells which leads to a labeling of F-actin. Such an experimental approach can bring major technical hurdles such as transfection efficiency and duration.

Non-toxic Actin fluorescent stains for live-cell imaging

Spirochrome recently released unique fluorescent stains for monitoring Actin (and Tubulin) easily in living cells. These fluorescent stains (SiR-Actin and SiR-Tubulin) are cell permeable compounds which stain F-actin (and microtubules, respectively) in living cells.

3D-SIM microscopy image of labeled Actin stress fibers in human primary dermal fibroblasts.

3D-SIM microscopy image of labeled Actin stress fibers in human primary dermal fibroblasts. Source: Spirochrome.

The benefits are obvious:

  • No transfection
  • No washing steps
  • No toxic effects
  • Excellent brightness
  • Far-red excitation & emission
  • Deep tissue penetration and minimal background
  • Multiple fluorescent stainings with other dyes possible

If you’re interested in more information about these stains, take a look at my recent post 2 new Actin and Tubulin live-cell imaging stains – without transfection!

What do you think about these products? Have you tried any of them already? Leave your questions or comments below!

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