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View additional product information for ReadyProbes™ F-Actin Labeling Reagents - FAQs (R37179, R37112, R37178, R37110)
24 product FAQs found
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.
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.
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.
Yes. ReadyProbes ActinRed and ActinGreen reagents are designed for storage between 15°C and 30°C. Store the reagents according to the product instructions and protect them from light.
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.
ReadyProbes ActinRed and ActinGreen reagents are ready-to-use. Add 2 drops, or 80 µL, per mL to the labeling solution.
Phalloidin binds noncovalently to a conserved cleft between adjacent F-actin subunits in a stoichiometric ratio of one phalloidin molecule per actin subunit.
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.
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.
This is not recommended. The ReadyProbes reagents were developed for imaging applications whereas the Ready Flow reagents were optimized for flow cytometry.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.