Alexa fluor 555 conjugated phalloidin

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Alexa Fluor 555-conjugated phalloidin is a fluorescent dye-labeled version of the natural toxin phalloidin. It is used to selectively label and visualize F-actin, the filamentous form of the actin protein, in fixed and permeabilized cells.

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36 protocols using alexa fluor 555 conjugated phalloidin

Ethanol Effects on Neuronal Cytoskeleton

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E15 cortical neurons grown on glass coverslips were fixed with 4% paraformaldehyde for 15 min after ethanol exposure on DIV 3. Cells were blocked with 5% BSA and 0.2% TritonX-100 for 1 h at room temperature and incubated with mouse anti-Tuj1 antibody (1:1000, Promega) at 4 °C overnight, followed by AlexaFluor 488-conjugated secondary antibodies and AlexaFluor 555-conjugated phalloidin (1:500, Invitrogen) for 1 h at room temperature. Hoechst 33342 (2 μg/ml, Molecular Probes) was used to visualize individual cell nuclei. Images were collected with a Zeiss LSM780 laser scanning confocal microscope (Confocal and Two-Photon Imaging Core, SUNY Upstate Medical University). The average F-actin signal density at distal neurite (5μm from the tip) was normalized to the tubulin immunosignal intensity and compared between H₂O or EtOH treated group.

Wang D., Enck J., Howell B.W, & Olson E.C. (2019). Ethanol exposure transiently elevates but persistently inhibits tyrosine kinase activity and impairs the growth of the nascent apical dendrite.. Molecular neurobiology, 56(8), 5749-5762.

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Immunofluorescence Staining of Cells

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Cells were either grown in glass-bottom dishes (35 mm Petri dish with 14 mm glass surface, #1.5, MatTek) or on glass cover slips (d = 24 mm, #1.5, Gerhard Menzel). They were washed shortly with PBS at 37°C and fixed for 3 min in methanol followed by a 30 s immersion in acetone at -22°C. In case of actin staining with Alexa Fluor® 555-conjugated phalloidin (Invitrogen) cells were fixed for 10 min in 4% (w/v) paraformaldehyde (Merck) in PBS (pH 7.2–7.4; adjusted with NaOH at ≈ 70°C) at room temperature and permeabilized with 0.2% (v/v) Triton™ X-100 (Sigma-Aldrich) in PBS for 3 min. Fixed cells were washed in PBS and blocked for 1 h in PBS containing 5% (w/v) bovine serum albumin (BSA; Sigma-Aldrich). Thereafter, cells were incubated with primary antibodies for 1 h, washed with PBS, and incubated with secondary antibodies for 40 min [antibodies were dissolved in PBS containing 1% (w/v) BSA]. Before mounting with Mowiol (Carl Roth) on glass slides (76x26 mm; R. Langenbrinc) cells were washed with PBS and mono-distilled H₂O. Samples were dried over night at 4°C and stored at the same temperature until usage (within two weeks).

Moch M., Windoffer R., Schwarz N., Pohl R., Omenzetter A., Schnakenberg U., Herb F., Chaisaowong K., Merhof D., Ramms L., Fabris G., Hoffmann B., Merkel R, & Leube R.E. (2016). Effects of Plectin Depletion on Keratin Network Dynamics and Organization. PLoS ONE, 11(3), e0149106.

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Immunofluorescence Staining and Quantification

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Cells were fixed with 4% paraformaldehyde (Electron Microscopy Sciences) for 15 min and then permeabilized with 0.1% Triton X-100 (Roche Applied Science) for 20 min at room temperature. Fixed cells were then incubated with 10% goat serum (Invitrogen) for 1 hr and then primary antibodies for 1 hr. Alexa Fluor 488 and 555 conjugated goat anti-mouse (or anti-rabbit) IgG secondary antibodies (Invitrogen) were used as secondary antibodies. Alexa Fluor 555 conjugated phalloidin (Invitrogen) and 4′,6-diamidino-2-phenylindole (DAPI; Invitrogen) were used for visualization of actin microfilaments and nucleus, respectively. Percentage of marker-positive cells was quantified with a custom-developed MATLAB program (MathWorks) based on a watershed segregation algorithm.

Chen W., Han S., Qian W., Weng S., Yang H., Sun Y., Villa-Diaz L.G., Krebsbach P.H, & Fu J. (2018). Nanotopography Regulates Motor Neuron Differentiation of Human Pluripotent Stem Cells. Nanoscale, 10(7), 3556-3565.

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Immunofluorescence Labeling for Protein Detection

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As described previously^{26 (link)}, 4% paraformaldehyde (Electron Microscopy Sciences) was used for cell fixation before permeabilization with 0.1% Triton X-100 (Roche Applied Science). Primary antibodies listed in Supplementary Table 2 were then used for protein detection. For immunolabeling, goat-anti mouse Alexa Fluor 488 and/or goat-anti rabbit Alexa Fluor 546 secondary antibodies were used. To label actin microfilaments, Alexa Fluor 555 conjugated phalloidin (Invitrogen) was used. Cells were stained with 4,6-diamidino-2- phenylindole (DAPI; Invitrogen) to visualize the nucleus.

Xue X., Sun Y., Resto-Irizarry A., Yuan Y., Aw Yong K.M., Zheng Y., Weng S., Shao Y., Chai Y., Studer L, & Fu J. (2018). Mechanics-guided embryonic patterning of neuroectoderm tissue from human pluripotent stem cells. Nature materials, 17(7), 633-641.

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Histochemical Analysis of Tentacle Structures

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Tentacle samples were rinsed in 0.2 µm-filtered sterile artificial seawater (SASW) (91 ), and then fixed in 4% paraformaldehyde in SASW for 30 min, rinsed with PBS + 0.1% Triton-X100 (PBST), and incubated with Alexa-Fluor-555-conjugated phalloidin (Invitrogen) diluted 1:250 and DAPI (ThermoFisher) diluted 1:10,000 in PBST overnight at 4 °C with gentle agitation. Following staining, samples were thoroughly washed in PBST, and then transitioned through a series of 15% and 30% sucrose in PBS and incubated in OCT embedding medium (Fisher Sci.) overnight at 4 °C. Samples were then snap-frozen on dry ice and sectioned on a cryostat (Leica). Sections were mounted and imaged on a Zeiss LSM-700 scanning confocal microscope.

Clarke D.N., Rose N.H., De Meulenaere E., Rosental B., Pearse J.S., Pearse V.B, & Deheyn D.D. (2024). Fluorescent proteins generate a genetic color polymorphism and counteract oxidative stress in intertidal sea anemones. Proceedings of the National Academy of Sciences of the United States of America, 121(11), e2317017121.

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F-actin Staining in Quarter-turn Explants

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The presence of F-actin in quarter-turn explants was demonstrated with Alexa® Fluor 555-conjugated phalloidin (Invitrogen, Carlsbad, CA, USA). F-actin is a cytoskeleton marker and used to stain extant cells in the quarter-turn explant. Fixed specimens were washed three times with PBS and incubated with phalloidin (diluted in PBS containing 0.05% Tween-20) for 60 minutes followed by three washing steps with PBS. Next, nuclei of the specimens were counterstained with DAPI (diluted 1:1000 in PBS) for 15 minutes followed by washing with PBS and covered with Roti®-Mount FluorCare and a cover glass.

Schomann T., Mezzanotte L., De Groot J.C., Rivolta M.N., Hendriks S.H., Frijns J.H, & Huisman M.A. (2017). Neuronal differentiation of hair-follicle-bulge-derived stem cells co-cultured with mouse cochlear modiolus explants. PLoS ONE, 12(10), e0187183.

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Cell Morphology Quantification using Immunofluorescence

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To fix samples, half of the media was removed from each sample, and an equivalent volume of fixative (4% paraformaldehyde and 5% sucrose in PBS) was added for 20 minutes at room temperature. Samples were permeabilized in 0.5% Triton X-100 and blocked in 10% normal goat serum (Invitrogen) before incubating with primary antibodies in blocking solution. Samples were washed and incubated with fluorescent secondary antibodies, 1:2000 Hoechst 33342 (Invitrogen, Thermo Fisher Scientific) and, if applicable, 1:100 AlexaFluor 555-conjugated phalloidin (Invitrogen, Thermo Fisher Scientific), and then washed before mounting on slides using ProLong Gold Antifade solution (Life Technologies, Thermo Fisher Scientific). To quantify cell morphology, phalloidin staining was analyzed using CellProfiler software [25] (link). In CellProfiler, Hoechst staining was used to identify nuclei, with a minimum diameter of 10 pixels, and shape was used to distinguish clumped objects. The phalloidin staining for each cell was propagated from the identified nuclei, and the area and axis lengths of each cell were determined.

Jones C.E., Hammer A.M., Cho Y., Sizemore G.M., Cukierman E., Yee L.D., Ghadiali S.N., Ostrowski M.C, & Leight J.L. (2018). Stromal PTEN Regulates Extracellular Matrix Organization in the Mammary Gland. Neoplasia (New York, N.Y.), 21(1), 132-145.

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Sucrose Effects on Cytoskeleton Visualization

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For confocal imaging, cells were seeded onto collagen-coated glass coverslips in 6 well plates at a density of 2 × 10⁵ cells per well. The following day, cells were exposed to 125, 250 or 750 mM sucrose for 10 or 30 minutes, or 24 hours. Cells were then washed with PBS, fixed with 4% paraformaldehyde, and permeabilized with 0.1% triton X-100 for 15 minutes. PBS containing 1% BSA was used as a blocking agent prior to incubation with 200 nM Alexa Fluor 555 conjugated phalloidin (Invitrogen, Carlsbad, CA, USA) and anti-tubulin FITC conjugated antibody (Abcam Inc., Cambridge, MA, USA). Coverslips were then washed and mounted on glass slides using Prolong® Gold Antifade Reagent with DAPI (Fisher Scientific, Grand Island, NY, USA). Samples were imaged using either a Nikon upright or inverted fluorescent microscope equipped with either a 20 or 40× dry objective.

Pulikkathara M., Mark C., Kumar N., Zaske A.M, & Serda R.E. (2017). Sucrose modulation of radiofrequency-induced heating rates and cell death. Convergent science physical oncology, 3(3), 035001.

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Quantifying Forskolin-Induced Cell Rounding

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Mouse Y1 cells and human adrenocortical tumor cells were treated with 10 mM forskolin for 30 min and the degree of cell rounding was estimated by counting cells displaying spherical shape in a given field, and expressing them as a percentage of the total cells in view [10, (link)26, 27] (link). Each field contained a minimum of 30 cells, and the observations were taken on five separate, random fields. The counting was performed by two independent operators. Y1 cells, transfected with wild type, S3A, S3D cofilin, or empty vector or silenced for cofilin were stimulated for 3 h with 10 mM forskolin, since time course experiments showed no effects of cell rounding before, and then analysed as described above.
For actin staining, Y1 cells and adrenocortical tumor cells were fixed with 4% paraformaldehyde for 10 min and stained with Alexa Fluor 555 conjugatedphalloidin (Invitrogen, Life Technologies Inc., Carlsbad, CA) for 20 min at room temperature. After wash with PBS, cells were mounted on glass slides with ProLong Diamond Antifade mounting medium (Life Technologies, Carlsbad, CA) and analysed by both fluorescence microscopy (Axio Vert.A1, Zeiss) and TCS SP2 laser scanning confocal microscope with a HeNe 543 nm laser and a 63 Â objective (HCX PL APO 63X/1.4e0.60 OIL) (Leica Microsystem, Deerfield, IL).

Peverelli E., Catalano R., Giardino E., Treppiedi D., Morelli V., Ronchi C.L., Vaczlavik A., Fusco N., Ferrero S., Bertherat J., Beuschlein F., Chiodini I., Arosio M., Spada A, & Mantovani G. (2017). Cofilin is a cAMP effector in mediating actin cytoskeleton reorganization and steroidogenesis in mouse and human adrenocortical tumor cells. Cancer letters, 406.

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Visualizing Lamellipodia Formation in Cells

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Cells were cultured on Matrigel (growth factor-reduced, BD Biosciences)-coated coverslips and serum-starved in low-serum (0.1% fetal bovine serum [FBS]) Dulbecco’s Modified Eagle’s Medium (DMEM) for 24 h. The cells were treated with either fluvoxamine or vehicle (0.1% DMSO) for 15 min and then stimulated with 10% FBS for 15 min to induce lamellipodia formation. The cells were washed with PBS, fixed with 4% paraformaldehyde for 10 min, and blocked with PBS containing 5% normal goat serum (Abcam) and 0.3% Triton X-100 for 30 min. G-actin and F-actin were stained with Alexa Fluor 488-conjugated DNase I (Life Technologies) and Alexa Fluor 555-conjugated phalloidin (Life Technologies), respectively.
For immunocytochemistry, cells were incubated with an anti-p-Y397-FAK antibody (44-624G, Life Technologies) for 1 h then incubated with Alexa Fluor 488-conjugated goat anti-rabbit IgG (Life Technologies) for 1 h. Coverslips were mounted with 50% glycerol, and confocal images were acquired using a confocal laser microscope (FV-300, Olympus).

Hayashi K., Michiue H., Yamada H., Takata K., Nakayama H., Wei F.Y., Fujimura A., Tazawa H., Asai A., Ogo N., Miyachi H., Nishiki T.I., Tomizawa K., Takei K, & Matsui H. (2016). Fluvoxamine, an anti-depressant, inhibits human glioblastoma invasion by disrupting actin polymerization. Scientific Reports, 6, 23372.

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