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Phalloidin staining protocol

Phalloidin staining protocol including buffers, reagents and a detailed procedure for staining with phalloidin dye conjugates.

Phalloidin is a highly selective bicyclic peptide that is used for staining actin filaments (also known as F-actin). It binds to all variants of actin filaments in many different species of animals and plants.

Typically, it is used conjugated to a fluorescent dye, such as FITC, Rhodamine, TRITC or similar dyes, such as Alexa Fluor® 488 or iFluor 488.

Phalloidin can be used with sample types such as formaldehyde-fixed and permeabilized tissue sections, cell cultures and cell-free experiments. It can also be used in paraffin-embedded samples that have been de-paraffinized.


Choosing the right phalloidin conjugate

A wide choice of phalloidin conjugates are available from several suppliers: the most popular are Phalloidin-FITC and Phalloidin-rhodamine.

We recommend our Phalloidin-iFluor dye conjugates, these are available as individual reagents and in a complete F-actin staining kit format. The iFluor dyes are brighter and more photostable than traditional dyes such as FITC and rhodamine and provide similar performance to Alexa Fluor® dyes.

Phalloidin conjugates available include
Phalloidin-iFluor 488
- Phalloidin-iFluor 405
-  Phalloidin-iFluor 555 
-  Phalloidin-iFluor 647

Unlabeled phalloidin can be used as a control in F-actin staining. As an alternative to dye conjugated phalloidin, biotin-conjugated phalloidin can be used with streptavidin-dye-conjugates.

Required reagents

The following reagents are required for this protocol:

  • Phalloidin conjugate – prepare following manufacturers guidelines, we recommend including 1% BSA to minimize the amount of phalloidin that binds to the tube.
  • PBS
  • Methanol-free formaldehyde
  • Triton X-100
  • Mounting media – we recommend  Fluoroshield (ab104135) to reduce bleaching, alternatively use PBS-buffered 50% glycerol with an anti-bleaching agent: p-phenylene diamine or N-propyl gallate
  • Optional: BSA, ethanolamine, glycine, DNA staining dye, lysopalmitoylphosphatidylcholine  

Preparing cell cultures of adherent cells

1 Grow cells in a 96 well black wall/clear bottom plate until they reach confluence (70–80%).

2 Cells can also be grown directly on coverslips inside a petri dish.

3 Aspirate cell culture medium (with care to avoid dislodging cells).

4 Wash once in PBS.

Preparing cell cultures of cells in suspension

1 Grow cells until they reach desired confluence (70–80%).

2 Centrifuge cells at 1,000 rpm for 5 minutes and aspirate the supernatant, preserving the cell pellet.

3 Resuspend the cell pellets gently in pre-warmed (37°C) growth medium and transfer to microplate or coverslips.

4 Aspirate cell culture medium carefully to avoid dislodging cells. Wash once in PBS.

Staining procedure for cultured cells

1 Fix cells in 3–4% formaldehyde in PBS at room temperature for 10–30 minutes.

2 Aspirate fixation solution and wash cells 2–3 times in PBS.

Optional: quench excess formaldehyde with 10 mM ethanolamine in PBS (or 0.1 M glycine in PBS) for 5 min.

Optional: add 0.1% Triton X-100 in PBS into the fixed cells for 3–5 minutes to increase permeability. Then wash cells 2–3 times in PBS.

3 Add phalloidin-conjugate working solution. Incubate at room temperature for 20–90 minutes.

Optional: add DNA staining dye at this point.

Optional: for vertebrate cells, it may be possible to add phalloidin-conjugate to the final PBS wash and mount in that medium.

4 Rinse cells 2–3 times with PBS, 5 min per wash

5 Add mounting media to preserve fluorescence (and seal to slide, if using coverslips)

6 Observe the cells at Ex/Em 493/517 nm

Procedure for other sample types

We recommend varying the fixation time and formaldehyde concentration across a range to optimize fixation conditions for staining.

For paraffin-embedded sections, we recommend deparaffinization following our protocol. Antigen retrieval is not necessary for phalloidin staining.

NB slides that have been fixed in 4% formalin may not preserve cytoskeletal structures effectively.

Tips for success

1 Phalloidin is pH sensitive: at elevated pH, a key thioether bridge is cleaved and the phalloidin loses its affinity for actin.

2 Phalloidin has an LD50 toxicity of 2mg/kg – handle according to good laboratory practice. However, the amounts used are typically low enough not to pose a significant critical risk.

3 Phalloidin staining can be combined with antibody-based staining. Add the phalloidin conjugate either during the primary or secondary antibody incubation step.

4 The optimal concentration of phalloidin conjugate and incubation time will vary depending on the particular cell type, fixation/sample prep conditions and/or permeability of the cells/ tissues to the probe.

5 Avoid fixatives containing methanol or acetone: these disrupt the structure of actin and prevent phalloidin staining.

6 Suspension cells can be attached to poly-D-lysine microplates or coverslips and then stained using the protocol for adherent cells.

7 Pre-incubating fixed cells with 1% BSA in PBS for 20–30 minutes may improve staining.

8 When staining coverslips, keep them in a covered container to minimize evaporation.

9 A fast one-step approach to phalloidin staining is effective in some circumstances: with a 20 minute incubation at 4ºC in 3.7% formaldehyde and 50–100 µg/mL lysopalmitoylphosphatidylcholine with phalloidin conjugate, followed by three washes and mounting.

10 If cells do not appear healthy add serum (2–10% range) to stain and wash solutions.

11 When used in unfixed samples, phalloidin binding leads to a decrease in the disassociation rate of actin subunits from the ends of actin filaments, essentially stabilizing actin filaments through the prevention of filament depolymerisation.

Typical images

Phalloidin Staining Image iFluor 488

Actin filament staining in HeLa cells. Actin filaments (green) were stained with Phalloidin-iFluor 488 reagent (ab176753); tubulin filaments were stained with a mouse anti-tubulin antibody/goat anti-mouse IgG (red). Nuclei were stained with Hoechst 33342.

Phalloidin staining image ab176755

Actin filament staining in HeLa cells. Actin filaments (red) were stained with Phalloidin-iFluor 555 reagent (ab176755). Nuclei were stained with Nuclear Green DCS1 (ab138905).

Phalloidin staining image ab176512

Actin filament staining in HeLa cells. Actin filaments (blue) were stained with Phalloidin-iFluor 350 reagent (ab176751).

Cy and CyDye are trademarks of GE Healthcare Limited.
Alexa Fluor® is a registered trademark of Life Technologies.

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