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Fluorescently labeled phalloidin

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Fluorescently labeled phalloidin is a compound used in microscopy to stain and visualize actin filaments within cells. It binds specifically to F-actin, allowing for the observation and analysis of the cellular cytoskeleton.

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Lab products found in correlation

Hoechst 33342, by Thermo Fisher Scientific (2 mentions) Hoescht dye, by Thermo Fisher Scientific (1 mentions) Chamber slide, by Ibidi (1 mentions) Eclipse ti e, by Nikon (1 mentions) Vinculin, by Abcam (1 mentions) Goat anti gfp, by Rockland Immunochemicals (1 mentions) Rabbit anti dsred, by Rockland Immunochemicals (1 mentions) Alexa dye, by Thermo Fisher Scientific (1 mentions) 4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions) Draq5, by Thermo Fisher Scientific (1 mentions) Rabbit anti β gal, by Thermo Fisher Scientific (1 mentions) Alexa fluor labelled secondary antibody, by Thermo Fisher Scientific (1 mentions) Lsm800, by Leica (1 mentions) Vectashield, by Vector Laboratories (1 mentions) Phosphate buffered saline (pbs), by Fujifilm (1 mentions) Uplan apo 60 oil objective lens, by Olympus (1 mentions) Fv1000, by Olympus (1 mentions) Appropriate secondary antibody, by Thermo Fisher Scientific (1 mentions) P akt, by Cell Signaling Technology (1 mentions) Hoechst 33258, by Merck Group (1 mentions)

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Phalloidins (2 citations)

8 protocols using fluorescently labeled phalloidin

1

Monocyte Morphology Imaging Protocol

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Sorted monocytes were imaged after being seeded in chamber slides (ibidi) and allowed to adhere. Cells were fixed in 4% paraformaldehyde, permeabilized with a saponin solution, and stained with Hoescht dye and fluorescently labeled phalloidin (both from Molecular Probes), and mAbs against β-tubulin (BD) and vinculin (Abcam). Cell morphology was captured on an Eclipse TiE epifluorescent microscope with NIS Elements deconvolution software (Nikon).
Boyette L.B., Macedo C., Hadi K., Elinoff B.D., Walters J.T., Ramaswami B., Chalasani G., Taboas J.M., Lakkis F.G, & Metes D.M. (2017). Phenotype, function, and differentiation potential of human monocyte subsets. PLoS ONE, 12(4), e0176460.
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2

Hemocyte Isolation and Immunostaining in Drosophila

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To analyze hemocytes ex vivo, hemocytes of single flies were released in 100μl of Schneider’s medium (Gibco, Millipore) or PBS supplemented with Complete 2x (Roche) and proteinase inhibitor 4-(2-Aminoethyl)benzenesulfonyl fluoride hydrochloride AEBSF (Sigma); flies were dissected and hemocytes were scraped from all inside areas of the fly (head, thorax and abdomen).
For immunohistochemistry, antibodies used were goat anti-GFP (Rockland Immunochemicals, 1:2000), rabbit anti-βGal (Thermo, 1:1000), rabbit anti-DsRed (Rockland Immunochemicals, 1:1000), mouse P1 (P1a+P1b) (Kurucz et al., 2007a (link)) (kind gift of I. Ando, 1:10), anti-Crq (Franc et al., 1996 (link)) (kind gift of C. Kocks, 1:1000), anti-Srp (kind gift of A. Giangrande, 1:1000) and secondary antibodies conjugated to Alexa dyes (Molecular Probes, 1:500), fluorescently labeled phalloidin (Molecular Probes), DAPI (Sigma), DRAQ5 (ThermoFisher).
Fat body cells were labeled using OilRedO (37%) dissolved in triethyl phosphate (6ml triethyl phosphate and 4 ml water) for 30 min, followed by three to four washes with distilled water. Other stainings used fluorescent LipidTOX dyes (LifeTech), diluted in PBS.
Bosch P.S., Makhijani K., Herboso L., Gold K.S., Baginsky R., Woodcock K.J., Alexander B., Kukar K., Corcoran S., Jacobs T., Ouyang D., Wong C., Ramond E.J., Rhiner C., Moreno E., Lemaitre B., Geissmann F, & Brückner K. (2019). Adult Drosophila lack hematopoiesis, but rely on a blood cell reservoir at the respiratory epithelia to relay infection signals to surrounding tissues. Developmental cell, 51(6), 787-803.e5.
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3

Immunostaining and RNA Hybridization Protocols

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Immunostaining and RNA in situ hybridizations were essentially performed as described in (Miller et al., 2017) . The following antibodies were used: mouse anti-P1/NimC1 (Kurucz et al., 2007) , mouse anti-Col (Krzemien et al., 2007) , mouse anti-ß integrin/Myospheroid (CF6.G11, DSHB), mouse anti-Antp (8C11, DSHB), rabbit anti-GFP (Torrey Pines), anti-DIG coupled to alkaline phosphatase (Roche) and Alexa-fluor labelled secondary antibodies (Molecular Probes). Nuclei were stained with DAPI and the actin cytoskeleton with fluorescently labeled phalloidin (Molecular Probes). The slides were mounted in Vectashield (Vector) and observed under a confocal microscope (Zeiss LSM800 or Leica SP5). Confocal images are displayed as maximum intensity projection of Z-stacks.
Boulet M., Renaud Y., Lapraz F., Benmimoun B., Vandel L., & Waltzer L. (2021). Characterization of the Drosophila adult hematopoietic system reveals a rare cell population with differentiation and proliferation potential.
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4

Immunofluorescence Staining of Cultured Cells

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Cells cultured on glass-bottom dishes were fixed with 4% paraformaldehyde in phosphate-buffered saline (Wako) at 37°C for 30 min, permeabilized with 0.1% Triton X-100 for 15 min, blocked with 5% normal goat serum for 1 h, and incubated with the primary antibody (Supplemental Table S3) for 1.5 h and then hybridized with appropriate secondary antibodies (Thermo Fischer Scientific) for 1 h at room temperature. The cell nucleus and actin filaments were stained with Hoechst 33342 (Thermo Fischer Scientific) and fluorescently labeled phalloidin (Thermo Fischer Scientific), respectively. Images were acquired using a microscope (IX73, Olympus) or a confocal laser scanning microscope (FV1000, Olympus) equipped with a UPlan Apo 60× oil objective lens (NA = 1.42) and analyzed using ImageJ software.
Liu S., Matsui T.S., Kang N, & Deguchi S. (2022). Analysis of senescence-responsive stress fiber proteome reveals reorganization of stress fibers mediated by elongation factor eEF2 in HFF-1 cells. Molecular Biology of the Cell, 33(1), ar10.
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5

Probing T Cell Activation Signaling

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CD4 and CD8 T cells were purified by negative selection and expanded for 48 hr using platebound anti-CD3 (clone 145–2c11; 1μg/mL) and anti-CD28 (clone 37.51; 1μg/mL). Cells were collected and allowed to rest on ice for 2 hr prior to stimulation with planar lipid bilayers as described above, for 15 min. Cells were subsequently fixed with 4% paraformaldehyde, permeabilized with 0.1% T-100, and blocked with PBS containing 2% bovine serum albumin and 5% goat serum before incubation with pZAP70 (cell signaling technologies, cat#2701), pERK (cell signaling technologies, cat#4370) or pAKT (cell signaling technologies, cat#4060) Abs, as well as fluorescently labeled phalloidin (ThermoFisher). All antibodies were used at 1:200. Images were acquired with a Marianas CSU-W with super resolution via optical reassignment (SoRa) spinning disc equipped with a 100X oil objective, Prime95B sCMOS camera and Slidebook software (Intelligent Imaging Innovations).
Guy C., Mitrea D.M., Chou P.C., Temirov J., Vignali K.M., Liu X., Zhang H., Kriwacki R., Bruchez M.P., Watkins S.C., Workman C.J, & Vignali D.A. (2022). LAG3 associates with TCR-CD3 complexes and suppresses signaling by driving co-receptor-Lck dissociation. Nature immunology, 23(5), 757-767.
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6

Immunofluorescence Microscopy of HS1 and Actin

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Immunofluorescence microscopy was carried out using standard techniques. At specified time points, cells were fixed with 4% paraformaldehyde for 10 minutes, then permeabilized with 0.1% Triton X-100 in PBS for 10 minutes. Samples were then blocked using 5% BSA for 2 hours at room temperature. Samples were stained with primary antibodies targeting HS1 (D5A9; Cell Signaling Technology), followed by appropriate secondary antibodies conjugated with Alexa fluorphores (Invitrogen). Cells were also stained for actin cytoskeleton using fluorescently-labeled phalloidin (Invitrogen) and nuclear stain Hoechst 33342 (Thermo).
Chaudhuri P.K., Wang M.S., Black C.T., Huse M, & Kam L.C. (2020). Modulating T cell activation using depth sensing topographic cues. Advanced biosystems, 4(9), e2000143.
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7

Immunofluorescence Staining of Cells

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Unless stated otherwise, 200,000 cells were plated on collagen-coated coverslips (18 mm, No. 1.5) in a six-well plate well and kept in a CO2 incubator for 48 h to form a freshly confluent monolayer. Cells were fixed either with ice-cold methanol for 1 min (for keratin staining) or with 4% PFA for 10 min (for F-actin staining). When PFA was used for fixation, cells were subsequently permeabilized with 0.2% Triton X-100 for 10 min. Nonspecific antigen interactions were blocked with 5% BSA in PBST for 1 h. Incubation with primary antibodies was performed either for 3 h at RT or overnight at 4°C in a humidified chamber. Coverslips were incubated with secondary antibodies, together with Hoechst 33258 (Sigma-Aldrich) and, when indicated, also with fluorescently labeled phalloidin (Invitrogen) for 1 h at RT in the dark.
Prechova M., Adamova Z., Schweizer A.L., Maninova M., Bauer A., Kah D., Meier-Menches S.M., Wiche G., Fabry B, & Gregor M. (2022). Plectin-mediated cytoskeletal crosstalk controls cell tension and cohesion in epithelial sheets. The Journal of Cell Biology, 221(3), e202105146.
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8

Immunoblotting Antibodies Catalogue Reference

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Antibodies used in this study were ABCC2/ MRP2 (catalogue number MAB4150; Millipore, Burlington, MA), RAB11A (mouse) (catalogue number 610656; BioScience, Franklin Lakes, NJ), RAB11A (rabbit) (catalogue number ab128913; Abcam, Cambridge, MA), myosin Vb (catalogue number NBP1-87746; Novus), FLAG (catalogue number F3165; Sigma-Aldrich, St Louis, MO), UNC45A (rabbit) (catalogue number HPA039228; Merck, Kenilworth, NJ), UNC45A (mouse) (catalogue number ADI-SRA-1800-F; Enzo Life Sciences, Farmington, NY), beta-tubulin (catalogue number T4026; Merck), c-myc (catalogue number 631206; Takara, Kusatsu, Japan), ezrin (catalogue number SC58758; Santa Cruz Biotechnology, Dallas, TX), beta-actin (catalogue number A5441; Sigma-Aldrich), syntaxin-3 (catalogue number ab133750; Abcam), and munc18-2 (catalogue number ab103976; Abcam). Fluorescently labeled phalloidin (catalogue number A22284) was from Invitrogen.
Li Q., Zhou Z., Sun Y., Sun C., Klappe K, & van IJzendoorn S.C. (2022). A Functional Relationship Between UNC45A and MYO5B Connects Two Rare Diseases With Shared Enteropathy. Cellular and Molecular Gastroenterology and Hepatology, 14(2), 295-310.
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