Fluorescent Phalloidin: A Practical Stain for Visualizing Actin Filaments

Actin is a highly conserved family of proteins that form microfilaments. Its amino acid sequence varies less than 20% between organisms as simple as yeast to higher eukaryotes such as humans. Due to its intracellular abundance in eukaryotic cells, well within micromolar concentrations, actin participates in the most protein-protein interactions than any other known protein. It possesses a unique ability to dynamically polymerize into filamentous actin (F-actin) from its monomeric state. Interactions between F-actin and regulatory proteins such as actin-binding proteins rapidly assemble and disassemble actin filaments organizing them into actin bundles and cytoskeletal networks. These higher-order structures provide the mechanical and structural support essential for a multitude of cellular processes including intracellular transport, cytokinesis, cell motility, polarity and cell shape, gene regulation and signal transduction. Because actin is essential in so many biological processes, tools (actin-specific antibody and phalloidin derived stains) for the visualization of these actin structures are essential in research.

Actin-Specific Antibodies: The Earliest F-Actin Stains

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The earliest actin staining technique adopted an immunohistochemical (IHC) approach by employing fluorophore labeled antibodies to stain against actin microfilaments (Lazarides and Weber, 1974). Although this approach functionally works for actin visualization, it did have disadvantages.

First, immunostaining is labor intensive. It requires careful consideration of parameters including antibody selection, fixation, antigen-retrieval, and blocking. If the desired antibody is not commercially available, then additional time and cost is needed to develop and purify the appropriate antibodies.

Second, the utilization of an antibody may be an obstacle. Antibodies, which are generally large in size (?150 kDa), are impermeable to cell membranes and require fixation and permeabilization of the cell prior to actin staining. Proper fixation is critical, and is dependent on epitope sensitivity and the antibody being used. Fixation preserves cell structures but it can reduce the antigenicity of certain cellular compartments. Therefore, it is imperative to optimize fixation conditions because under or over-fixation can drastically reduce or prohibit tissue immunoreactivity.

Lastly, the non-specific binding of antibody conjugates to both monomeric and polymeric actin generates high levels of background noise which results in low resolution stains. To combat such limitations, novel actin-binding components with enhanced specificity for F-actin were developed as suitable alternatives, the most notable being phalloidin.

Phalloidin

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Phalloidin, the main representative of the phallotoxin family, is a bicyclic heptapeptide isolated from the poisonous death cap mushroom, Amanita phalloides. It possesses a high binding affinity for the grooves between F-actin subunits over monomeric G-actin. Once bound to F-actin, phalloidin shifts the equilibrium of monomers and filaments toward the filaments position, and inhibits ATP-hydrolysis. The interaction stabilizes actin filaments by preventing subunit dissociation, and it promotes actin polymerization by lowering the critical concentration. As a consequence, these characteristics have made phalloidin derivatives useful stains in visualizing F-actin and cytoskeletal networks.

Properties of Phalloidin Derivatives

Since its initial use to stain actin in the late 1970s, phalloidin and its fluorescent derivatives remain the gold standard for visualizing cellular actin filaments. Compared to IHC staining, phalloidin derivatives offer significant advantages for labeling actin. They provide a convenient method for identifying, labeling, and quantitating F-actin formation in fixed and permeabilized cells and tissue sections.

Phalloidin derivatives are water soluble and when used at nanomolar concentrations can selectively stain F-actin. Unlike some antibodies against actin, phalloidin's capacity to bind more tightly to actin filaments over monomers decreases non-specific staining and background noise which produce high contrast between stained and unstained areas. In comparison to antibodies, phalloidin derivatives' are small (<2 kDa) and phalloidin bound filaments do not impede functional properties of the filaments. The small size also permits for denser F-actin labeling producing more detailed stains when imaged at higher resolutions. In addition, because actin is evolutionarily conserved, the binding properties of phalloidin derivatives can be utilized in staining a wide range of animal and plant cells.

Choosing the Right Phalloidin Derivative

Due to its notable staining abilities, an array of phalloidin derivatives has been developed to study and visualize actin filaments and actin-related structures. Summaries of the properties of unlabeled, biotinylated, and fluorescently labeled phalloidin derivatives can be found below.

Unlabeled Phalloidin

Unlabeled phalloidin has limited uses. Other than serving as promotor of actin polymerization, unlabeled phalloidin is typically used as a control in blocking F-actin staining.

Biotinylated Phalloidin

Biotinylated phalloidin provides and indirect method for visualizing and quantitating actin filaments. It requires the use of a fluorescent or enzyme-conjugated avidin or streptavidin for visualization. Whilst indirect detection offers the benefit of signal amplification, the need for additional reagents increases costs and assay time. Additionally, when staining with biotinylated phalloidin a higher concentration of the phalloidin conjugate is required, approximately two-fold more than needed with fluorescent phalloidin. Regardless of the inconveniences, biotinylated phalloidin are favorable for pull-down and immunoprecipitation assays for investigating protein interactions and colocalization with F-actin.

Fluorescent Phalloidin-iFluor^® Conjugates

Exploiting the excellent fluorescence properties of our iFluor^® dyes, AAT Bioquest has developed a series of phalloidin conjugates spanning the full color spectrum which selectively bind to F-actins. Used at nanomolar concentration, these iFluor^® phalloidin derivatives are convenient for many actin-related assays. Such assays include labeling, identifying, and quantitating F-actins in formaldehyde-fixed and permeabilized tissue sections, cell-cultures, or cell-free experiments. Compared to traditional fluorescein isothiocynanate (FITC) and rhodamine conjugates, Phalloidin-iFluor^® conjugates offer F-actin stains that are superior in brightness and photostability. Phalloidin-iFluor^® conjugates are equivalent, and in most cases superior, to Alexa Flour^® conjugates in performance. Finally, Phalloidin-iFluor^® conjugates are more affordable as they are available in a unit size of 300 tests for $145 versus Alexa Fluor^®'s $433 price tag.

We encourage the research community to try our Phalloidin-iFluor^® conjugates with their respective protocols for a more intense fluorescence in their F-actin staining. Phalloidin-iFluor^® 700, iFluor^®-750 and iFluor^®-790 conjugates are among the few near-infrared stains available with sufficient spectral separation from commonly used red fluorophores.

 

Staining F-actin with Phalloidin-iFluor^® Conjugates

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The following is a sample protocol for staining f-actin in cultured cells utilizing AAT Bioquest Phalloidin-iFluor^® 488 conjugate (Cat No. 23115).

Storage & Handling Conditions

It is important to note that phalloidin is toxic, although the contents present in each vial are potentially lethal to a mosquito (LD₅₀ of phalloidin = 2 mg/kg), it should always be handled with care.

Phalloidin-iFluor^® 350 to Phalloidin-iFluor^® 596 conjugates come packaged as 1000X stock solution in DMSO and are ready to be used as supplied. Phalloidin-iFluor^® 633 to Phalloidin-iFluor^® 790 conjugates come packaged as a lyophilized powder and must be prepared in 1000X stock solution. Both conjugates are stable for at least six months when stored at proper conditions. If not to be used immediately, store Phalloidin-iFluor^® conjugate at -20 °C, protect from light and avoid freeze-thaw cycles.

Materials

• Phalloidin-iFluor^® 488 conjugate
• PBS
• 3.0 - 4.0% Methanol-free formaldehyde
• 0.1% Triton X-100
• BSA
• Cell culture or tissue sample for staining
• Optional: ethanolamine
• Optional: FluoroQuest Mounting Medium with DAPI

Actin Staining Protocol

Note: If using paraffin-embedded tissue samples, a deparaffinization and rehydration step is required. For a deparaffinization and rehydration protocol see Appendix A.

1. Warm the vial of Phalloidin-iFluor^® 488 to room temperature and centrifuge briefly before opening.
2. Prepare a 1000X Phalloidin-iFluor^® 488 DMSO stock solution by adding 30 µL of DMSO into the vial of Phalloidin-iFluor^® 488.
3. Prepare a 1X Phalloidin-iFluor^® 488 working solution by adding 1 µL of 1000X Phalloidin-iFluor^® 488 conjugate DMSO solution to 1 mL of PBS with 1% BSA. Mix well by pipetting solution up and down. This makes enough staining solution for 10 wells at 100 µL/well. Aliquot any unused 1000X Phalloidin-iFluor^® 488 DMSO stock solution and store under proper conditions.
   • Note: Staining may vary between different cell types. For optimal performance, the concentration of phalloidin conjugate working solution should be prepared accordingly.
4. Fix cells in 3.0 - 4.0% methanol-free formaldehyde in PBS at room temperature for 10 to 30 minutes.
5. Aspirate fixation solution carefully.
6. Rinse fixed cells 2 to 3 times in PBS
7. Optional: Quench excess formaldehyde with 10 mM ethanolamine in PBS for 5 minutes.
8. Increase permeability by adding 0.1% Triton X-100 in PBS into fixed cells for 3 to 5 minutes. Rinse cells 2 to 3 times in PBS.
9. In a 96-well plate, add 100 µL/well of Phalloidin-iFluor^® 488 working solution into fixed cells. Stain cells at room temperature for 20 to 90 minutes.
10. Rinse cells gently 2 to 3 times with PBS to remove excess phalloidin conjugate.
11. Optional: Add a small drop of FluoroQuest Mounting Medium with DAPI and incubate for 2 hours. This inhibits photobleaching and counterstain nucleus with DAPI.
12. Observe cells by using a fluorescence microscope equipped with the appropriate filter set. Phalloidin-iFluor^® 488 requires a FITC filter set for visualization.

Appendix

Deparaffinization and Re-Hydration of Paraffin-Embedded Tissue Slides:

A deparaffinization step is only required when using paraffin-embedded tissue sections. Prior to F-actin staining, it is important to de-paraffinize and rehydrate your tissue slides. Incomplete removal of paraffin will result in poorly or non-stained sections.

It is important to note that phalloidin is not compatible with methanol fixation. Please use methanol-free formaldehyde or glutaraldehyde to fix tissue samples.

Materials and Reagents

• Xylene
• 100% ethanol
• 95% ethanol
• 70% ethanol
• 50% ethanol

Method

1. Heat tissues slides at 55°C for 10 minutes to melt paraffin.
2. Deparaffinize tissue slides in 2 changes of xylene or xylene substitute for 5 minutes each.
3. Gradually hydrate tissue slides by transferring slides through 100%, 95% and 70% alcohol for 5 minutes each.
4. Rinse in PBS 2X for 5 minutes each.
5. Proceed with F-actin staining protocol.

References

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1. Dominguez, Roberto, and Kenneth C. Holmes. Actin Structure and Function. Annual review of biophysics40 (2011): 169"186.PMC. Web. 19 Mar. 2018.
2. Firat-Karalar, Elif Nur, and Matthew D. Welch. New Mechanisms and Functions of Actin Nucleation. Current opinion in cell biology23.1 (2011): 4"13.PMC. Web. 19 Mar. 2018.
3. Lazarides, Elias, and Klaus Weber. Actin Antibody: The Specific Visualization of Actin Filaments in Non-Muscle Cells. Proceedings of the National Academy of Sciences of the United States of America71.6 (1974): 2268"2272. Print.
4. Lengsfeld, Anneliese M. et al. Interaction of Phalloidin with Actin. Proceedings of the National Academy of Sciences of the United States of America71.7 (1974): 2803"2807. Print.
5. Melak, Michael, et al. Actin Visualization at a Glance. Development, vol. 144, no. 4, 2017, doi:10.1242/dev.150052.
6. Wulf, E et al. Fluorescent Phallotoxin, a Tool for the Visualization of Cellular Actin. Proceedings of the National Academy of Sciences of the United States of America76.9 (1979): 4498"4502. Print.

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