Alexa fluor 594 conjugated phalloidin

Manufactured by Thermo Fisher Scientific

Sourced in United States, United Kingdom

Alexa Fluor 594-conjugated phalloidin is a fluorescent probe used for the detection and visualization of filamentous actin (F-actin) in cells. It binds specifically to F-actin, allowing for the labeling and imaging of the actin cytoskeleton.

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Alexa Fluor 594- and 488-conjugated phalloidin (2 citations) Alexa Fluor 488- or 594-conjugated phalloidin (2 citations) Alexa 594-conjugated Phalloidin (19 citations) Alexa Fluor–conjugated phalloidin (44 citations) Alexa 594-phalloidin (29 citations) Alexa Fluor phalloidin (43 citations) Alexa Fluor 594 (2045 citations) Phalloidin 594 (24 citations) Alexa 594 (538 citations) Alexa Fluors (21 citations)

80 protocols using alexa fluor 594 conjugated phalloidin

Immunostaining of Caco-2 and Tissue Sections

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Caco-2_BBe monolayers grown on 5 cm² Transwell supports were fixed in 2% paraformaldehyde and immunostained as described previously. Snap-frozen tissues were stored at −80°C, after which 5 μm frozen sections were cut and fixed in 1% paraformaldehyde. EdU was detected using the Alexa Fluor 488 click chemistry detection kit (Invitrogen). Immunostaining used affinity-purified rabbit anti-human MLCK1 (2 μg/ml),^{7 (link)} monoclonal mouse anti-MLCK clone K36 (Sigma-Aldrich, 1 μg/ml)^{7 (link)}, affinity-purified rabbit anti-pMLC (Cell Signaling 3671, 1 μg/ml), monoclonal mouse anti-occludin clone OC-3F10 (Invitrogen, 1 μg/ml), monoclonal mouse anti-E-cadherin clone M168 (Abcam, 2 μg/ml), or monoclonal rat anti-CD3 clone CD3–12 (Abcam, 2 μg/ml). Primary antibodies were followed by Alexa Fluor 488- or Alexa Fluor 594-conjugated affinity-purified secondary antibodies (Invitrogen) along with Alexa Fluor 594-conjugated phalloidin (Invitrogen) and Hoechst 33342 (Invitrogen). Stained sections were mounted in Prolong gold (Invitrogen).

Graham W.V., He W., Marchiando A.M., Zha J., Singh G., Li H.S., Biswas A., Ong M.L., Jiang Z.H., Choi W., Zuccola H., Wang Y., Griffith J., Wu J., Rosenberg H.J., Wang Y., Snapper S.B., Ostrov D., Meredith S.C., Miller L.W, & Turner J.R. (2019). Intracellular MLCK1 diversion reverses barrier loss to restore mucosal homeostasis. Nature medicine, 25(4), 690-700.

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Immunostaining of Caco-2 and Tissue Sections

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Comprehensive Immunofluorescence Analysis of Gut-on-a-Chip

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For immunofluorescence microscopic analysis, the cells grown in the gut-on-a-chip were fixed with 4% (wt/vol) paraformaldehyde for 15 min, washed twice for 5 min with 0.1% bovine serum albumin (BSA) in phosphate buffer saline (PBS), and then permeabilized with 0.2% (vol/vol) Triton X-100 (Sigma Aldrich, MO, USA) for 20 min. After washing with 0.1% BSA in PBS, the cells were incubated with 3% (wt/vol) BSA blocking solution for 1 h. Subsequently, the cells were incubated with primary antibodies overnight at 4 °C, washed three times, incubated with secondary antibodies for 90 min, and washed three times with 0.1% BSA in PBS. The following antibodies were used for immunohistochemistry: mouse anti-ZO-1 (Invitrogen, USA, 1:200), rabbit anti-PECAM (Abcam, 1:500), mouse anti-MUC2 (Invitrogen, USA, 1:500), Alexa Fluor 594-conjugated phalloidin (Invitrogen, USA, 1:250), Alexa Fluor 488-conjugated Wheat Germ Agglutinin (Invitrogen, USA, 5 μg/mL, 1:200), Goat anti-mouse Alexa Fluor 488 (Invitrogen, USA, 1:1000), and Donkey anti-rabbit Alexa Fluor 594 (Invitrogen, USA, 1:1000). Samples were then incubated with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI; Molecular Probe, OR, USA) to visualize cell nuclei before taking confocal microscopic images (Olympus, Japan).

Jeon M.S., Choi Y.Y., Mo S.J., Ha J.H., Lee Y.S., Lee H.U., Park S.D., Shim J.J., Lee J.L, & Chung B.G. (2022). Contributions of the microbiome to intestinal inflammation in a gut-on-a-chip. Nano Convergence, 9, 8.

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Immunostaining and FISH for Intestinal Tissues

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Small and large intestine were dissected and fixed overnight in 1.6% paraformaldehyde (Thermo Scientific) containing 20% sucrose at 4 °C. Samples were then placed in OCT (Tissue-Tek) and snap-frozen over dry ice. Tissue sections of 8-mm thickness were cut, air-dried and blocked using blocking solution. Tissues were then labelled using an Alexa Fluor 594-conjugated phalloidin (Invitrogen) or a primary mouse anti-mouse pan-cytokeratin antibody (clone PCK-26) (Abcam) for 60 min in a humidified atmosphere followed by a secondary goat anti-mouse Alexa Fluor 594 (Thermo Fisher Scientific) for 30 min, then stained with DAPI, and mounted using Fluoromount-G. For fluorescent in situ hybridization, small intestine and large intestine were dissected and prepared as described for frozen sections^{33 (link)}. Following tissue blocking, sections were incubated with 0.45 pmol μl⁻¹ eubacterial oligonucleotide probe (AminoC6 +Alexa Fluor 594) 5′-GCTGCCTCCCGTAGGAGT-3′; (Operon)^{33 (link)} in a pre-chilled hybridization buffer (Sigma) overnight at 4 °C. Sections were counterstained with DAPI and mounted with Fluoromount-G.

Cummings R.J., Barbet G., Bongers G., Hartmann B.M., Gettler K., Muniz L., Furtado G.C., Cho J., Lira S.A, & Blander J.M. (2016). Different tissue phagocytes sample apoptotic cells to direct distinct homeostasis programs. Nature, 539(7630), 565-569.

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BMSCs Cytoskeleton Visualization

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After 1 h of seeding onto the prepared matrices, BMSCs were fixed in 4% formaldehyde, stained by Alexa Fluor 594-conjugated phalloidin (Invitrogen), and enclosed in VECTASHIELD medium with 4',6-diamidino-2-phenylindole (DAPI) (Vector Laboratories, Burlingame, CA, USA).

Ida T., Kaku M., Kitami M., Terajima M., Rosales Rocabado J.M., Akiba Y., Nagasawa M., Yamauchi M, & Uoshima K. (2018). Extracellular matrix with defective collagen cross-linking affects the differentiation of bone cells. PLoS ONE, 13(9), e0204306.

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Quantifying TGF-β1-induced Actin Polarization

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KO1, KO2, and EV cells were seeded into μ‐Slide I IbiTreat chambers (no. 80106; Ibidi, Planegg, Germany), which have reservoirs for medium on both ends. After incubation for 12 hours to allow adherence, the medium was exchanged for DMEM without FBS, and the cells were incubated for 12 hours. Subsequently, recombinant TGF‐β1 (R&D Systems, Minneapolis, MN, USA) was added to the left reservoir (20 ng), and the cells were incubated for 4 hours. Next, the cells were fixed and subjected to F‐actin staining using Alexa Fluor 594‐conjugated phalloidin (Invitrogen, Carlsbad, CA, USA). Fluorescence signals were visualized using the LSM710 laser scanning microscope (Carl Zeiss, Oberkochen, Germany). Cells in which F‐actin staining was concentrated in the direction of TGF‐β1 were enumerated in 10 random fields at high magnification, and the proportion among the total number of cells was calculated.

Tahara S., Nojima S., Ohshima K., Hori Y., Kurashige M., Wada N., Motoyama Y., Okuzaki D., Ikeda J, & Morii E. (2019). Serum deprivation‐response protein regulates aldehyde dehydrogenase 1 through integrin‐linked kinase signaling in endometrioid carcinoma cells. Cancer Science, 110(5), 1804-1813.

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Visualizing Actin Cytoskeleton in Saos-2 Cells

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For further cell adhesion examination, cell cultures were rinsed twice with PBS and fixed with 4% paraformaldehyde in PBS for 30 min at RT, then rinsed twice in PBS. Cells were permeabilized with 0.1% triton X-100 in PBS for 15 min at RT. Actin filaments were stained by incubating the samples with Alexa Fluor 594-conjugated phalloidin (Invitrogen, ThermoFisher Scientific, Waltham, MA, USA), and the cell nucleus was stained with Hoechst 33,258 (Sigma-Aldrich, Saint Louis, MO, USA) for 15 min at RT in the dark. Saos-2 actin distribution was observed under a confocal microscope (Confocal TCS SP5, Leica, Wetzlar, Germany).

Careta O., Salicio-Paz A., Pellicer E., Ibáñez E., Fornell J., García-Lecina E., Sort J, & Nogués C. (2021). Electroless Palladium-Coated Polymer Scaffolds for Electrical Stimulation of Osteoblast-Like Saos-2 Cells. International Journal of Molecular Sciences, 22(2), 528.

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Actin Polymerization in Macrophages

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Actin polymerization was performed as described previously [6] (link). Briefly, macrophages were plated in 96-well plates. The cells were fixed with 4% paraformaldehyde for 20 min, washed, and permeabilized with 0.1% Triton X-100. The cells were washed and stained with Alexa Fluor 594-conjugated phalloidin (Invitrogen) and then stained with 4′,6-diamidino-2-phenylindole (DAPI). Actin polymerization was measured as a relative fluorescent unit of phalloidin to DAPI.

Xin C., Quan H., Kim J.M., Hur Y.H., Shin J.Y., Bae H.B, & Choi J.I. (2018). Ginsenoside Rb1 increases macrophage phagocytosis through p38 mitogen-activated protein kinase/Akt pathway. Journal of Ginseng Research, 43(3), 394-401.

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Analyzing Cell Adhesion on Alloy Surfaces

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Cell adhesion onto the alloy surface was analysed using an antibody against vinculin to determine the presence of focal contacts. At the same time, phalloidin was used to visualize actin filaments and their distribution, as reported previously [8 (link)]. The same cell culture protocol described for viability studies was employed, but after 24 h of culture the cells were fixed in 4% PFA in PBS for 30 min at RT, permeabilised with 0.1% Triton X-100 (Sigma) in PBS for 15 min and blocked for 25 min with 1% bovine serum albumin (BSA; Sigma) in PBS at RT. Samples were then incubated with 2 μg/ml mouse anti-vinculin primary monoclonal-antibody (Chemicon, MAB3574) for 60 min at RT and washed with 1% BSA-PBS. Then, samples were incubated with a mixture of 1.4 U/ml Alexa fluor 594-conjugated phalloidin (Invitrogen), 6 μg/ml Alexa fluor 488 goat anti-mouse IgG1 and Hoechst 33258 (both from Sigma) for 60 min at RT. Finally, samples were washed in 1% BSA-PBS, air dried and mounted on specific bottom glass dishes (MatTek) using ProLong mounting solution (Life Technologies). Control analyses were performed in absence of the alloy. Sample evaluation was done with a confocal laser scanning microscope (CLSM, Olympus). One disk was analysed for each surface modification and alloy composition.

Blanquer A., Hynowska A., Nogués C., Ibáñez E., Sort J., Baró M.D., Özkale B., Pané S., Pellicer E, & Barrios L. (2016). Effect of Surface Modifications of Ti40Zr10Cu38Pd12 Bulk Metallic Glass and Ti-6Al-4V Alloy on Human Osteoblasts In Vitro Biocompatibility. PLoS ONE, 11(5), e0156644.

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Assessing Tat-Induced Integrin and Actin Dynamics

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U937 cells (6×10⁴) were pretreated with Tat in the presence or absence of cannabinoid (1 μM; THC, CP55940; 2h at 37°C), then seeded in Lab-Tek Chamber Slides (Nunc, ThermoFisher Scientific, Waltham, MA) precoated with Coll IV (20 μg/ml) and incubated at 37°C (1h) to allow for cellular adhesion. The cells were fixed in 4% paraformaldehyde (1h) and rinsed in PBS. Cells were permeabilized with 0.1% Triton X-100 in PBS (20 min) then blocked with 5% BSA in PBS (1h). Immunofluorescent staining was performed using a FITC-conjugated anti-human β₁- integrin antibody (1:250, 2h)(Millipore, Billerica, MA), followed by AlexaFluor594-conjugated Phalloidin to visualize F-actin (1:500, 2h)(Invitrogen). Cells were counterstained with DAPI (1:20,000, 5 min)(Invitrogen) for nuclear identification. ProLong Gold Antifade Reagent (Invitrogen) was used for mounting of the coverslips in addition to stabilization of the immunofluorescent signal. Image acquisition was performed using a BX51 microscope with a spinning disk unit (Olympus, Center Valley, PA) and an Orca-R2 CCD camera (Hamamatsu, Japan). Images then were processed using the Slidebook software package (Intelligent Imaging Innovations, Denver, CO, USA).

Raborn E.S., Jamerson M., Marciano-Cabral F, & Cabral G.A. (2014). Cannabinoid inhibits HIV-1 Tat-stimulated adhesion of human monocyte-like cells to extracellular matrix proteins. Life sciences, 104(0), 15-23.

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