Invitrogen™ Phalloidin Labeling Probes

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.

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.

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).

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.

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.

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

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.

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.

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.

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.

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.

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

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

No. They differ only in the dyes attached to the phalloidin. The phalloidin has the same structure in all of the products.

No. The dye in Alexa Fluor Plus 405 Phalloidin is structurally different from Alexa Fluor 405 dye and has distinct spectral properties; Alexa Fluor 405 dye has excitation/emission maxima of 402/421 nm; Alexa Fluor Plus 405 Phalloidin has excitation/emission maxima of 405/450 nm.