Actin Staining Protocol

We offer several fluorescent and biotinylated phalloidin and phallacidin derivatives for labeling F-actin. These phallotoxins, isolated from the deadly Amanita phalloides mushroom,¹ are bicyclic peptides that differ by two amino acid residues. They can be used interchangeably in most applications and bind competitively to the same sites in F-actin. Phalloidin and phallacidin contain an unusual thioether bridge between a cysteine and tryptophan residue that forms an inner ring structure. At elevated pH, this thioether is cleaved and the toxin loses its affinity for actin.

Fluorescent and biotinylated phallotoxins stain F-actin at nanomolar concentrations and are extremely water soluble, thus providing convenient probes for labeling, identifying, and quantitating F-actin in tissue sections, cell cultures, or cell-free experiments.^1-3 Labeled phallotoxins have similar affinity for both large and small filaments, binding in a stoichiometric ratio of about one phallotoxin molecule per actin subunit in muscle and nonmuscle cells from many different species of plants and animals. Unlike antibodies, the binding affinity does not change appreciably with actin from different species or sources. Nonspecific staining is negligible, and the contrast between stained and unstained areas is extremely large. It has been reported that phallotoxins are unable to bind to monomeric G-actin.¹ Phallotoxins shift the monomer/polymer equilibrium toward the polymer, lowering the critical concentration for polymerization up to 30-fold.^3,4 Phallotoxins also stabilize F-actin, inhibiting depolymerization by cytochalasins, potassium iodide, and elevated temperatures.

Because the phallotoxin conjugates are small, with an approximate diameter of 12–15 Å and molecular weight of <2000 daltons, a variety of actin-binding proteins—including myosin, tropomyosin, troponin, and DNase I—can still bind to actin after treatment with phallotoxins. Even more significantly, phallotoxin-labeled actin filaments remain functional; labeled glycerinated muscle fibers still contract, and labeled actin filaments still move on solid-phase myosin substrates.^6,7 Fluorescent phallotoxins can also be used to quantitate the amount of F-actin in cells.^8,9 The unlabeled phallotoxins may be used as controls in blocking F-actin staining or in promoting actin polymerization. Our biotin-XX phalloidin allows researchers
to visualize actin filaments by electron microscopy using standard enzyme-mediated avidin/streptavidin techniques. Eosin phalloidin can potentially be used for correlated fluorescence and electron microscopic studies. Deerinck and co-workers reported that when eosin conjugates are excited in the presence of diaminobenzidine (DAB), an insoluble, electron-dense DAB oxidation product is formed that is easily visualized with either light or electron microscopy.¹⁰

Even though phallotoxins have an LD₅₀ of approximately 2 mg/kg when injected into the mouse, the toxins of A. phalloides that are responsible for most of the symptoms and fatalities associated with poisoning by this mushroom are the structurally related amatoxins. The major in vivo lesions produced by injected phallotoxin occur in the liver and are associated with stabilization of polymeric actin. Phallotoxins, however, tend not to be absorbed by the gastrointestinal tract even though they are stable between pH 3 and pH 9.

Storage and Safety
Once reconstituted in methanol, the stock solutions are stable for at least one year when stored frozen at =–20°C, desiccated, and protected from light. It appears that NBD phallacidin, and possibly all of these toxins, exhibit a small loss of activity when stored in aqueous solution at 2–6°C for over three weeks.

While the amount of toxin present in a vial could be lethal only to a mosquito (LD₅₀ of phalloidin = 2 mg/kg), it should be handled with care.

Materials Required but Not Provided

• Methanol
• PBS
• Methanol-free formaldehyde
• Acetone or Triton X-100
• Fluorescent or enzyme-conjugated streptavidin, as prepared in step 1.9, for use with biotin-XX phalloidin only
• BSA, optional
• Image-iT FX Signal Enhancer (I36933), optional
• lysopalmitoylphosphatidylcholine, optional
  

Preparing the Stock Solution

Fluorescent Phallotoxins

The vial contents should be dissolved in 1.5 mL methanol to yield a final concentration of 200 units/mL, which is equivalent to approximately 6.6 µM.

One unit of phallotoxin is defined as the amount of material used to stain one microscope slide of fixed cells, according to the following protocol (see step 1.6), and is equivalent to 5 µL of methanolic stock solution for the fluorescent phallotoxins.

Biotin-XX Phalloidin

Staining cells with biotin-XX phalloidin (B7474) requires 1) the use of a higher concentration of the phallotoxin conjugate than when staining with fluorescent phallotoxins and 2) the addition of a fluorescent or enzyme-conjugated avidin or streptavidin detection reagent (see step 1.9). The vial contents should be dissolved in 0.5 mL methanol to yield a final concentration of 100 units/mL, which is equivalent to approximately 20 µM.

For biotin-XX phalloidin, 10 µL of the methanolic stock solution is equivalent to one unit of phallotoxin, which is defined as the amount of material used to stain one microscope slide of fixed cells according to the following protocol (see step 1.6).

Unlabeled Phalloidin

Solutions of this product should be prepared just like the fluorescent phallotoxins described in Fluorescent Phallotoxins, taking into account the larger quantity of material provided.

The procedure below was originally developed for use with NBD phallacidin.¹¹ It has been successfully used with all of Invitrogen Molecular Probes phallotoxin conjugates. This procedure may not be optimum for a particular experimental system, but has yielded consistent results in most instances. The following protocol describes the staining procedure for adherent cells grown on glass coverslips.

1.  Formaldehyde-Fixed Cells

1. Wash cells twice with prewarmed phosphate-buffered saline, pH 7.4 (PBS).


2. Fix the sample in 3.7% formaldehyde solution in PBS for 10 minutes at room temperature. Note: Methanol can disrupt actin during the fixation process. Therefore, it is best to avoid any methanol containing fixatives. The preferred fixative is methanol-free formaldehyde.


3. Wash two or more times with PBS.


4. Place each coverslip in a glass petri dish and extract it with a solution of acetone at =–20°C or 0.1% Triton X-100 in PBS for 3 to 5 minutes.


5. Wash two or more times with PBS.


6. When staining with any of the fluorescent phallotoxins, dilute 5 µL methanolic stock solution into 200 µL PBS for each coverslip to be stained. To reduce nonspecific background staining with these conjugates, add 1% bovine serum albumin (BSA) to the staining solution. It may also be useful to pre-incubate fixed cells with PBS containing 1% BSA or with the Image-iT FX signal enhancer (I36933) for 20–30 minutes prior to adding the phallotoxin staining solution. When staining with biotin-XX phalloidin (B7474), dilute 10 µL of the methanolic stock solution into 200 µL PBS for each coverslip to be stained. When staining more than one coverslip, adjust volumes accordingly. For a stronger signal, use 2 or 3 units per coverslip.


7. Place the staining solution on the coverslip for 20 minutes at room temperature (generally, any temperature between 4°C and 37°C is suitable). To avoid evaporation, keep the coverslips inside a covered container during the incubation.


8. Wash two or more times with PBS.


9. When using biotin-XX phalloidin, incubate for 30 minutes with 100 µL of a 10 µg/mL solution of fluorescent or enzyme-conjugated streptavidin dissolved in 100 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.3% Triton X-100 and 1% BSA. Incubate for 15 minutes at room temperature. After incubation, wash the coverslip with PBS. To develop enzyme activity, follow a procedure recommended for the specific enzyme.


10. For long-term storage, the cells should be air dried and then mounted in a permanent mountant such as ProLong Gold reagent or Cytoseal. Specimens prepared in this manner retain actin staining for at least six months when stored in the dark at 2–6°C.

2.  Simultaneous Fixation, Permeabilization, and Fluorescent Phallotoxin Staining

The phallotoxins appear to be stable for short periods in 4% formaldehyde fixation buffers. This permits a rapid one-step fixation, permeabilization, and labeling procedure as follows.

1. Prepare a 1 mL solution containing 50 to 100 µg/mL lysopalmitoylphosphatidylcholine and 3.7% formaldehyde and then add 5–10 units of fluorescent phallotoxin (approximately 25 to 50 µL of methanolic stock solution).


2. Place this staining solution on cells and incubate for 20 minutes at 4°C.


3. Rapidly wash three times with buffer.


4. Mount coverslips and view.

Photostability or resistance to photobleaching is a primary concern when making fluorescence measurements. Invitrogen brand product lines such as Alexa Fluor, Oregon Green, BODIPY, and rhodamine fluorophores (including Texas Red™-X) are significantly more photostable than NBD and fluorescein and will therefore enable more accurate photographic measurements.

To further reduce photobleaching, minimize the exposure of fluorescently labeled specimens to light with neutral density filters and expose samples only when observing or recording a signal. Maximize collection of fluorescence by using a minimum of optics, high–numerical aperture objectives, relatively low magnification, high-quality optical filters, and high-speed film or high-efficiency detectors.

Invitrogen Antifade reagents, including Molecular Probes SlowFade Gold and ProLong Gold antifade reagents, can extend the useful lives of many fluorescent probes. These reagents are also available in a formulation with DAPI. They can be used on fixed cell preparations but are not compatible with living cells. Cytoseal also appears to protect BODIPY fluorophores from photobleaching.

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