Phalloidin staining protocol
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
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.
- 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
Grow cells in a 96-well black wall/clear bottom plate until they reach confluence (70–80%).
Aspirate cell culture medium (with care to avoid dislodging cells).
Wash once in PBS.
Avoid fixatives containing methanol or acetone: these disrupt the actin structure and prevent phalloidin staining.
Suspension cells can be attached to poly-D-lysine microplates or coverslips and then stained using the protocol for adherent 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
Grow cells until they reach desired confluence (70–80%).
Centrifuge cells at 1,000 rpm for 5 minutes and aspirate the supernatant, preserving the cell pellet.
Resuspend the cell pellets gently in pre-warmed (37°C) growth medium and transfer to microplate or coverslips.
Aspirate cell culture medium carefully to avoid dislodging cells.
- 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
Fix cells in 3–4% formaldehyde in PBS at room temperature for 10–30 minutes.
Pre-incubating fixed cells with 1% BSA in PBS for 20–30 minutes may improve staining.
When staining coverslips, keep them in a covered container to minimize evaporation.
Aspirate fixation solution and wash cells 2–3 times in PBS.
Quench excess formaldehyde with 10 mM ethanolamine in PBS (or 0.1 M glycine in PBS) for 5 min..
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.
If cells do not appear healthy, add serum (2–10% range) to stain and wash solutions.
Rinse cells 2–3 times with PBS, 5 min per wash.
Add mounting media to preserve fluorescence (and seal to the slide if using coverslips).
Observe the cells at Ex/Em 493/517 nm.
- The below image illustrates 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.