Invitrogen™ Phalloidin Labeling Probes

Product Code.	Color	Excitation Wavelength Range	Dye Type	unitSize
10174712	Blue	346/442 nm	Alexa Fluor™ 350	Each
15932941	Violet	405/450 nm	Alexa Fluor™ Plus 405	Each
10125092	Green	495/518 nm	Alexa Fluor™ 488	300 units
10730355	Green	496/520 nm	Oregon Green™ 488	Each
10615963	Green	496/516 nm	FITC (Fluorescein)	Each
10184712	Yellow	531/554 nm	Alexa Fluor™ 532	Each
10063052	Red-orange	540/565 nm	TRITC	300 units
10634053	Orange	556/570 nm	Alexa Fluor™ 546	Each
10389132	Orange	555/565 nm	Alexa Fluor™ 555	Each
16628105	Orange	555/565 nm	Alexa Fluor Plus 555	50µg
10643853	Red	558/569 nm	BODIPY™	Each
10135092	Orange-red	578/600 nm	Alexa Fluor™ 568	Each
10751144	Red	581/609 nm	Alexa Fluor™ 594	Each
10164852	Red	591/608 nm	Texas Red™	Each
10053252	Far-red	632/647 nm	Alexa Fluor™ 633	5g
10124892	Far-red	633/647 nm	Alexa Fluor™ 635	Each
10656353	Far-red	650/668 nm	Alexa Fluor™ 647	5g
16400815	Far-red	650/668 nm	Alexa Fluor Plus 647	Pack of 300
10402322	Near-infrared	663/690 nm	Alexa Fluor™ 660	Each
10656163	Near-infrared	679/702 nm	Alexa Fluor™ 680	Each
16316512	Near-infrared	758/784 nm	Alexa Fluor™ Plus 750	Each
10125002	None	None	Biotin-XX	Each
10718853	None	None	Phalloidin (unlabeled)	1mg

Product Code. 10174712 Supplier Invitrogen™ Supplier No. A22281

Description

Achieve precise and reliable F-actin staining with fluorescent and biotinylated phalloidins. Phalloidin conjugates are widely used in imaging applications to selectively label F-actin in a variety of sample types including fixed and permeabilized cells, tissue sections, and cell-free experiments.

Fluorescent and biotinylated phalloidins are water soluble and bind to filamentous actin (F-actin) with nanomolar affinity, making them convenient probes for labeling, identifying, and quantifying F-actin in cryopreserved tissue sections, fixed and permeabilized cells, and cell-free experiments. Phalloidin conjugates bind similarly to actin from various species, including plants and animals, enabling staining of the cytoskeleton in a wide range of samples.

A variety of phalloidin conjugates for filamentous (F-actin) staining are available, including fluorescent Alexa Fluor and Alexa Fluor Plus phalloidins, along with phalloidins conjugated to classic fluorescent dyes such as BODIPY, fluorescein, and rhodamine. Phalloidin staining is spectrally compatible with other fluorescent stains used in cellular analyses such as GFP/RFP, Qdot nanocrystals, and other Alexa Fluor conjugates and antibodies. Biotin‐XX Phalloidin can be used to visualize actin filaments via fluorescent streptavidin tags or standard enzyme-mediated avidin/streptavidin techniques such as in electron microscopy. Unlabeled phalloidin is available for use as a control in blocking F‐actin staining or in promoting polymerization.

Phalloidin conjugates bind to both large and small actin filaments with similar affinity in a 1:1 stoichiometry between phallotoxin and actin subunits. They do not bind G-actin monomers.

Alexa Fluor and Alexa Fluor Plus phalloidin conjugates for F-actin staining

Fluorescent Alexa Fluor dye conjugates of phalloidin are popular F-actin stains, offering color choices across the full spectral range. These phalloidin conjugates provide researchers with fluorescent probes that are superior in brightness and photostability compared to other spectrally similar conjugates.

Alexa Fluor Plus Phalloidin conjugates retain the same specificity for actin but offer 3-5 times greater sensitivity and brightness compared to the corresponding Alexa Fluor Phalloidin conjugate. This increased brightness is beneficial for challenging F-actin imaging, such as the super‐resolution microscopy methods SIM and STORM, and for reliable staining of actin stress fibers.

Features of phalloidin probes

• High specificity—binds selectively to F-actin, which allows for precise labeling of actin filaments in fixed cells and cryopreserved tissues
• Strong affinity—nanomolar binding affinity for F-actin, which ensures stable and reliable actin staining
• Extensive fluorescent conjugate options—over twenty conjugated varieties of phalloidin
• Compatibility with fixed samples—typically used with fixed cells and tissues, making them suitable for actin staining in detailed structural studies, immunofluorescence staining, and IHC applications
• Multiplexing capability—the wide availability of phalloidin conjugates enables their use in combination with other fluorescent probes and antibodies for multiplex imaging. Biotinylated phalloidin can be made use of in downstream streptavidin steps.
• Quantitative analysis—can be used for quantitative analysis of F-actin distribution and density within cells, aiding in the study of cytoskeletal dynamics. The unlabeled phalloidin can be titrated as a control.
• Ease of use—staining is straightforward and quick
• Excellent stability—exhibit good photostability, which is essential for prolonged imaging sessions and time-lapse studies
• Wide applicability—used for a range of applications, including studying cell morphology, motility, and the effects of drugs on the actin cytoskeleton

Order Info

Shipping Condition: Room Temperature

Specifications

• Color: Blue
• Content And Storage: Store in freezer -5°C to -30°C and protect from light.
• Excitation Wavelength Range: 346/442 nm
• Dye Type: Alexa Fluor™ 350
• For Use With (Equipment): Fluorescence Microscope, Flow Cytometer, Confocal Microscope, Compatible with DAPI filter set
• Quantity: 300 Units
• Product Line: Alexa Fluor
• Shipping Condition: Room Temperature
• Label Type: Alexa Fluor Dyes
• Product Type: Phalloidin
• Sub Cellular Localization: Actin, Cytoskeleton

Frequently Asked Questions (FAQs)

Do you offer alternative package sizes for phalloidin or ReadyProbes reagents?

Alternative package sizes may be available through a custom request process. Contact Thermo Fisher Scientific customer support or your sales representative to discuss available options for the specific reagent and quantity needed.

Can I use conjugated phalloidin label probes on organoids?

Yes. Conjugated phalloidin labeling probes can be used with organoids when F-actin is accessible after fixation and permeabilization. Because organoids are thicker than monolayer cell cultures, protocol optimization may be needed, including permeabilization conditions, probe incubation time, and probe concentration.

Are conjugated phalloidin reagents antibodies?

No, Phalloidin is not an antibody. It is a bicyclic peptide toxin originally isolated from the Amanita phalloides mushroom and is used to bind and label F-actin in fixed and permeabilized samples.

Can I use methanol instead of DMSO to reconstitute the conjugated phalloidins from the lyophilized solids?

Yes, methanol can be used to reconstitute fluorescent phalloidin, but it creates a less concentrated stock solution than DMSO. Dissolve the vial contents in 1.5 mL of methanol to yield a 40X stock solution at a concentration of 200 assays/mL, which is equivalent to approximately 6.6 µM. One unit/assay of fluorescent phalloidins is equivalent to 5 µL of the methanolic stock solution. Methanol may be used as a stock-solution solvent, but methanol-containing fixatives are not recommended because they can disrupt F-actin.

How do I reconstitute fluorescent conjugated phalloidin probes to make a stock solution?

For fluorescent phalloidin label probes, prepare a DMSO stock solution by dissolving the vial contents in 150 µL anhydrous DMSO to make a 400X stock, with a concentration of approximately 66 µM or 2,000 assays per mL. Store between -25°C and -5°C, protected from light. One unit/assay of fluorescent phalloidins is equivalent to 0.5 µL of the DMSO stock solution.

Can I use conjugated phalloidin reagents with IHC, FFPE sections, or cryosections?

Yes. Fluorescent phalloidin can be used with appropriately prepared fixed and permeabilized tissue sections, especially cryosections. FFPE processing and deparaffinization can include organic solvents such as xylene or acetone, which may alter F-actin and reduce phalloidin binding. If paraffin sections show weak or no signal, consider cryosections or an anti-actin antibody.

How long can I store samples labeled with fluorescent phalloidin reagents?

Image samples soon after staining unless they are mounted in a compatible antifade mountant. For longer-term storage, use an aqueous, curing antifade mountant that is compatible with the fluorophore, protect samples from light, and store at the temperature recommended in the product instructions. Avoid mountants containing organic solvents because they may affect F-actin labeling.

Which fluorescent phalloidin conjugate should I choose?

Choose the conjugate based on your microscope filters or lasers, other fluorophores in the experiment, and the signal intensity required. For example, avoid choosing an Alexa Fluor 488 phalloidin conjugate probe if your sample already expresses GFP or the experiment uses fluorescein conjugated antibodies. The user guide lists phalloidin conjugates across blue, green, orange/red, far-red, and near-IR channels. Alexa Fluor Plus conjugates are dyes that have been optimized for application-specific performance compared to classic Alexa Fluor conjugates for the same emission wavelength. Alexa Fluor conjugates are generally greatly improved compared to the classic fluorophore conjugate counterparts (i.e., Alexa Fluor 488 vs Fluorescein, Alexa Fluor 594 vs Texas Red).

Can I use these fluorescent phalloidin reagents in flow cytometry?

Yes. Fluorescent phalloidin reagents can be used in flow cytometry with fixed and permeabilized cells. Because flow cytometers and sample types vary, optimize the staining concentration, fixation, permeabilization, and acquisition settings for the instrument and sample. Include appropriate controls and compare samples processed in parallel.

Why might I see weak or no signal with conjugated phalloidin staining?

Weak or absent fluorescent signal can result from disrupted F-actin, the use of methanol-containing fixatives, insufficient permeabilization, competition for F-actin binding, incorrect stock solution preparation, low staining concentration, photobleaching, or imaging with the wrong filter set. When troubleshooting, start by using fresh methanol-free 4% paraformaldehyde for fixation, 0.1% Triton X-100 in PBS for permeabilization, and the recommended staining concentration for the conjugated phalloidin probe. Be sure to match the appropriate excitation/emission filter settings listed for the phalloidin conjugate in the user guide.

Do the conjugated phalloidin or ReadyProbes F-actin reagents covalently label cytoskeleton?

No. Phalloidin binds F-actin noncovalently with high affinity. It labels filamentous actin but does not form a covalent bond with the cytoskeleton.

What does a unit “U” mean in the 300 U quantity?

One unit is the amount of phalloidin used to label one microscope slide or coverslip of fixed cells. A 300 U vial contains enough lyophilized fluorescent phalloidin reagent for approximately 300 slides or coverslips when used according to the recommended protocol.

How long is the labeling step for the conjugated phalloidins?

For fluorescent phalloidin conjugates, incubate fixed and permeabilized cells with staining solution for 30-60 min at room temperature. For Biotin-XX phalloidin, incubate for 15 min at room temperature. Place the coverslips in a covered container to prevent evaporation during the incubation.

How should I store reconstituted phalloidin stock solution?

Reconstituted fluorescent phalloidin DMSO stock solution can be stored between -25°C and -5°C for up to 12 months when protected from light. If more than five freeze-thaw cycles are expected, aliquot the stock solution into smaller volumes before storage.

How many milligrams are in each conjugated phalloidin probe?

Phalloidin vial content is specified in units rather than milligrams because the approximate mass depends on the specific conjugate. Fluorescent phalloidin vials are listed as 300 U, and Biotin-XX phalloidin vials are listed as 50 U. The approximate molecular weight (MW) of unlabeled phalloidin is 790 Daltons (refer to the user guide for the approximate molecular weight of each conjugate). Depending on the fluorescent conjugate, there is approximately between 0.5 mg to 1 mg per vial.

What dilution of conjugated phalloidin probes should I use to label fixed mammalian cells?

For fluorescent phalloidin prepared as a 400X DMSO stock solution, dilute 0.5 µL of stock solution in 200 µL of PBS per coverslip. For fluorescent phalloidin prepared as a 40X methanol stock solution, dilute 5 µL of stock solution in 200 µL of PBS per coverslip. For Biotin-XX phalloidin, dilute 10 µL of methanol stock solution in 200 µL of PBS per coverslip.

Where do the conjugated phalloidins and ReadyProbes F-actin reagents bind on the F-actin in the cell?

Phalloidin binds noncovalently to a conserved cleft between adjacent F-actin subunits in a stoichiometric ratio of one phalloidin molecule per actin subunit.

The phalloidin vial looks empty. Is that normal?

Yes. The amount of lyophilized phalloidin conjugate is generally under 1 mg and may not be visible in the vial. Reconstitute using the volume specified in the user instructions, mix thoroughly and protect the stock solution from light.

What fixative should I use with mammalian cells prior to labeling with conjugated phalloidin probes?

We recommend fixing mammalian cells with methanol-free 4% formaldehyde or paraformaldehyde (PFA), followed by permeabilization with 0.1% Triton X-100 in PBS. Methanol-containing fixatives can disrupt F-actin and may reduce phalloidin binding.

Can phalloidin reagents be used across species or sample types?

Yes, fluorescent phalloidin conjugates can be widely used for F-actin staining in fixed and permeabilized mammalian cells, tissue cryosections, plants, and other eukaryotic samples. The phalloidin binding can still depend on sample preparation and accessibility, so yeast, fungi, thick tissues, organoids, or whole mounts may require optimized permeabilization, incubation time, or probe concentrations.

How is phalloidin labeling different from antibody labeling of actin?

Phalloidin binds directly to F-actin, while antibody labeling depends on antibody recognition of actin epitopes. Phalloidin has similar affinity for small and large actin filaments and is widely used across eukaryotic sample types when F-actin is accessible after fixation and permeabilization.

What do conjugated phalloidin reagents and ReadyProbes F-actin reagents label in the cell?

Conjugated phalloidin and ReadyProbes F-actin reagents label filamentous actin, or F-actin. They do not label monomeric G-actin. These reagents are commonly used to visualize the actin cytoskeleton in fixed and permeabilized samples.

Can I combine phalloidin or ReadyProbes F-actin staining with immunofluorescence or antibody staining?

Yes. Conjugated phalloidin and ReadyProbes F-actin staining can be combined with immunofluorescence, ICC, or antibody staining in fixed and permeabilized samples. A common workflow is to complete primary antibody staining first, then add phalloidin with the fluorescent secondary antibody or as a separate counterstaining step. Choose fluorophores with minimal spectral overlap.

Which fluorescent phalloidin reagent is used in the Cell Painting protocol?

The Cell Painting protocol uses Alexa Fluor 568 phalloidin. This reagent is available as a lyophilized fluorescent phalloidin conjugate and in a ready-to-use ReadyProbes ActinRed 568 reagent format.

What are the stock concentrations of conjugated phalloidin probes?

Fluorescent phalloidin conjugates are approximately 66 µM when prepared as a 400X DMSO stock solution and approximately 6.6 µM when prepared as a 40X methanol stock solution. Biotin-XX phalloidin is approximately 20 µM when prepared as a methanol stock solution.

Can I label live cells with conjugated phalloidin reagents?

Conjugated phalloidin and ReadyProbes F-actin reagents are used with fixed and permeabilized cells, not routine live-cell staining. Phalloidin conjugates are not typically cell permeant, and specialized delivery methods may affect actin organization or cell behavior. For live-cell actin imaging, consult the literature to find the staining experiment suitable for your experiments or consider a live-cell-compatible actin labeling approach.

Which phalloidin and ReadyProbes F-actin labeling reagents are available?

ReadyProbes F-actin reagents are available in ActinRed and ActinGreen formats, and phalloidin labeling probes are available as fluorescent conjugates, Biotin-XX conjugated phalloidin, and unconjugated phalloidin. Fluorescent dye options include Alexa Fluor dye conjugates, Alexa Fluor Plus dye conjugates, fluorescein, Oregon Green dye, rhodamine, BODIPY dye, Texas Red-X dye, and other visible to near-IR dye conjugates.

Can I use DAPI, NucBlue, SYTOX, or another nuclear counterstain with phalloidin or ReadyProbes F-actin reagents?

Yes. For fixed and permeabilized cells, spectrally compatible nuclear counterstains such as DAPI, NucBlue Fixed Cell reagent, or SYTOX reagents can be used with conjugated phalloidin or ReadyProbes ActinRed and ActinGreen reagents. Choose a nuclear counterstain that does not overlap with the fluorophore used for F-actin labeling.

Do I need to permeabilize cells prior to labeling with conjugated phalloidin and ReadyProbes F-actin reagents?

Yes, permeabilization after fixation is a required step for optimal labeling with conjugated phalloidin or ReadyProbes ActinRed and ActinGreen reagents. For mammalian cells, we recommend permeabilizing the sample in 0.1% Triton X-100 in PBS for 15 minutes followed by two or more PBS washes. Alternatively, if required, samples can be permeabilized in an acetone solution at or below 20°C in a glass petri dish.

How can I reduce nonspecific background when staining with conjugated phalloidin and ReadyProbes F-actin reagents?

Adding 1% w/v bovine serum albumin (BSA) to PBS wash buffer can help reduce nonspecific background when staining fixed and permeabilized cells with conjugated phalloidin or ReadyProbes ActinRed and ActinGreen reagents. After fixation, permeabilization, and PBS washes, wash 1-3 times with PBS containing 1% BSA or preincubate cells in PBS containing 1% BSA for 20-30 min before labeling. After labeling, wash 2-3 times with PBS before imaging.

Can I combine Click-iT or Click-iT Plus reactions with phalloidin conjugates used for actin staining?

We do not recommend using phalloidin conjugates for staining actin in combination with traditional Click-iT or Click-iT Plus reactions since phalloidin is extremely sensitive to the presence of copper.

For staining actin in combination with traditional Click-iT or Click-iT Plus reactions, we recommend using anti-α-actin antibodies for staining actin in the cytoskeleton. You can find a list of our actin antibodies here.

Another option would be to use the Click-iT Plus Alexa Fluor Picolyl Azide Toolkit (Cat. Nos. C10641, C10642, C10643). These Click-iT Plus toolkits provide Copper and Copper protectant separately which makes it easier to titrate the copper concentration to obtain optimal labeling with minimal copper-mediated damage. You may need to optimize the click reaction with the lowest possible concentration of copper and then perform the phalloidin staining.

I'm trying to label my paraffin sections for F-actin with a phalloidin conjugate, but I'm not seeing any signal. Why?

When cells and tissues are treated with solvents such as xylene or acetone (for example during deparaffinization of tissue sections), it affects the F-actin in a way that prevents phalloidins from binding. Phalloidin may be used with cryosections, which are not typically washed with organic solvents, or anti-actin antibodies may be used.

For Research Use Only. Not for use in diagnostic procedures.