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Phalloidin staining

Detailled procedure for staining with phalloidin dye conjugates, including tips for choosing the most suitable phalloidin conjugate.
Last edited Thu 18 Nov 2021

Phalloidin is a highly selective bicyclic peptide 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, phalloidin 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. 

Importantly, phalloidin is also pH sensitive: at elevated pH, a key thioether bridge is cleaved, and the phalloidin loses its affinity for actin. Phalloidin staining can be combined with antibody-based staining by adding the phalloidin conjugate during the primary or secondary antibody incubation step.

Stage 1 - Choosing the most suitable phalloidin conjugate

Materials required

  • Phalloidin conjugate – prepare following manufacturers guidelines
    • Tip: We recommend using 1% BSA solution to minimize the amount of phalloidin that binds to the tube.
    • Tip: 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.
  • PBS
  • Methanol-free formaldehyde
  • Optional: Triton X-100
  • Mounting media – we recommend Fluorosheild to reduce bleaching. Alternatively, use PBS-buffered 50% glycerol with an anti-bleaching agent: p-phenylenediamine or N-propyl gallate
  • Optional: BSA, ethanolamine, glycine, DNA staining dye, lysopalmitoylphosphatidylcholine  

Steps

1

Choosing the most suitable phalloidin conjugate.

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

Unlabelled 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.

ConjugateRecommended AbID
Palloidin-iFluor 350ab176751
Phalloidin-iFluor 488ab176753
Phalloidin-iFluor 405ab176752
Phalloidin-iFluor 555ab176756
Phalloidin-iFluor 594ab176757
Phalloidin-iFluor 647ab176759
Rhodamine Phalloidinab235138
Phalloidin FITCab235137

View our full range of Phalloidin-iFluor products

Stage 2 - Preparing culture of cells

Materials required

  • Phalloidin conjugate – prepare following manufacturers guidelines
    • Tip: We recommend using 1% BSA solution to minimize the amount of phalloidin that binds to the tube.
    • Tip: 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.
  • PBS
  • Methanol-free formaldehyde
  • Optional: Triton X-100
  • Mounting media – we recommend Fluorosheild to reduce bleaching. Alternatively, use PBS-buffered 50% glycerol with an anti-bleaching agent: p-phenylenediamine or N-propyl gallate
  • Optional: BSA, ethanolamine, glycine, DNA staining dye, lysopalmitoylphosphatidylcholine  

Steps

1

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

2

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

3

Wash once in PBS.

Stage 3 - Stain cultured cells with phalloidin conjugates

Materials required

  • Phalloidin conjugate – prepare following manufacturers guidelines
    • Tip: We recommend using 1% BSA solution to minimize the amount of phalloidin that binds to the tube.
    • Tip: 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.
  • PBS
  • Methanol-free formaldehyde
  • Optional: Triton X-100
  • Mounting media – we recommend Fluorosheild to reduce bleaching. Alternatively, use PBS-buffered 50% glycerol with an anti-bleaching agent: p-phenylenediamine or N-propyl gallate
  • Optional: BSA, ethanolamine, glycine, DNA staining dye, lysopalmitoylphosphatidylcholine  

Steps

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.

3

Add phalloidin-conjugate working solution.

  •  Incubate at room temperature for 20–90 minutes.
4

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

5

Add mounting media to preserve fluorescence (and seal to the slide if using coverslips).

6

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

Image 1 (above): 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.