To measure cell viability, the samples were stained with a live/dead kit (ThermoFisher) for 15 min at 37 °C. Cell morphology was assessed via f-actin/nuclei staining. After fixation with 4% paraformaldehyde (Electron Microscopy Sciences), f-actin and nuclei staining was performed using Alexa 594-phalloidin (ThermoFisher) and 4′,6-diamidino-2-phenylindole (DAPI, ThermoFisher), respectively, according to manufacturer’s instructions. Images were taken using a fluorescence microscope (Zeiss Axio Observer D1). At least five images were taken per sample and analyzed using ImageJ.
Alexa 594 phalloidin
Manufactured by Thermo Fisher Scientific
Sourced in United States
Alexa Fluor 594-phalloidin is a fluorescent dye that binds to F-actin, a structural component of the cytoskeleton in eukaryotic cells. It can be used to visualize and study the distribution and organization of actin filaments in fixed and permeabilized cells.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 710, by Zeiss (6 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (4 mentions)
Prolong gold antifade solution, by Thermo Fisher Scientific (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Lsm 510 meta, by Zeiss (2 mentions)
Hoescht 33342, by Merck Group (2 mentions)
Axioskop 2 plus, by Zeiss (2 mentions)
Live dead kit, by Thermo Fisher Scientific (1 mentions)
Paraformaldehyde (pfa), by Electron Microscopy Sciences (1 mentions)
Axio observer d1, by Zeiss (1 mentions)
Ab6672, by Abcam (1 mentions)
Anti flag, by Merck Group (1 mentions)
Prism 7, by GraphPad (1 mentions)
Sp8 confocal microscope, by Leica (1 mentions)
Ab52903, by Abcam (1 mentions)
Donkey anti rabbit alexa488, by Jackson ImmunoResearch (1 mentions)
Efficient navigation zen , by Zeiss (1 mentions)
Semi dry transfer apparatus, by Bio-Rad (1 mentions)
Vimentin, by Cell Signaling Technology (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
29 protocols using alexa 594 phalloidin
1
Cell Viability and Morphology Assay
2
Immunocytochemical Analysis of IL-6 in ARPE-19 Cells
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ARPE-19 cells were fixed with 4% paraformaldehyde in 100 mM phosphate buffered saline (PBS) for 15 min. After washing, cells were permeabilized and autofluorescence quenched in PBS containing 20 mM glycine, 75 mM ammonium chloride and 0.1% Triton X-100 for 8 min, followed by a 2 min incubation with this solution plus 0.5% sodium dodecyl sulfate. Cells were blocked in 0.25% fish skin gelatin, 0.025% saponin and 10% donkey serum in PBS for 1 h at 25 °C, followed by 5 min in high salt PBS. Cells were incubated overnight in a rabbit primary polyclonal antibody against IL-6 (Abcam, Cambridge, MA, USA; #ab6672, 1:500 in blocking solution) at 4 °C. After washing, cells were incubated for 1 h donkey anti-rabbit Alexa 488 (Abcam, 1:1000, in blocking solution) and F-actin counterstain (Alexa-594 phalloidin, 1:1000, Thermo Fisher Inc.) at 25 °C. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI; 1:10,000, 1 min). Antibody-antigen binding was fixed for 15 min in 4% paraformaldehyde. Staining was visualized using a Nikon Eclipse E600 epifluorescence microscope, and separate channels combined using Adobe Photoshop (San Jose, CA, USA) according to accepted protocols.
3
Immunofluorescence Imaging of Drosophila IFM
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Half thoraces were glycerinated (20 mM Na-Phosphate pH 7.2, 2 mM MgCl2, 2 mM EGTA, 5 mM DTT, 0.5% Triton X-100, 50% glycerol) overnight at -20°C. IFMs were dissected, washed and then fixed with 4% paraformaldehyde in relaxing solution (20 mM Na-Phosphate pH 7.2, 2 mM MgCl2, 2 mM EGTA, 5 mM DTT, 5 mM ATP) with protease inhibitors. Incubations of primary antibodies and Alexa 594-phalloidin (ThermoFisher Scientific) were carried out overnight at 4°C, followed by secondary antibody incubation for 3 hours at room temperature. Primary antibodies used were rat anti-α-actinin MAC276 (1:100, Babraham Bioscience Technologies), rat anti-Kettin KIg16 MAC155 (1:200, Babraham Bioscience Technologies), and anti-FLAG (1:500, Sigma-Aldrich). Fluorescently labeled secondary antibodies of the Alexa series (ThermoFisher Scientific) were used at a 1:400 dilution. Samples were mounted in ProLong Gold antifade solution (ThermoFisher Scientific).
Images were acquired on a LSM 510 Meta laser scanning confocal microscope using a 63x 1.4 NA Plan Apo oil immersion objective (Carl Zeiss).
Images were acquired on a LSM 510 Meta laser scanning confocal microscope using a 63x 1.4 NA Plan Apo oil immersion objective (Carl Zeiss).
4
Fluorescent Imaging of Indirect Flight Muscles
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Half thoraces were glycerinated (20 mM Na-Phosphate pH 7.2, 2 mM MgCl2, 2 mM EGTA, 5 mM DTT, 0.5% Triton X-100, 50% glycerol) overnight at -20°C. Indirect flight muscles were dissected, washed and then fixed with 4% paraformaldehyde in relaxing solution (20 mM Na-Phosphate pH 7.2, 2 mM MgCl2, 2 mM EGTA, 5 mM DTT, 5 mM ATP) with protease inhibitors. The incubation of rat anti-α-actinin antibody MAC276 (1:100; Babraham Bioscience Technologies) together with Alexa 594-phalloidin (1:100; ThermoFisher Scientific) was carried out overnight at 4°C, followed by secondary antibody incubation for 3 hours at room temperature. Fluorescently labeled secondary antibody of the Alexa series (ThermoFisher Scientific) was used at a 1:400 dilution. Samples were mounted in ProLong Gold antifade solution (ThermoFisher Scientific). All images were acquired using a 63x 1.4 NA HC Plan Apochromat oil objective on a Leica SP8 confocal microscope. One-way ANOVA followed by Tukey’s multiple mean difference post hoc tests were performed to determine statistically significant differences between genotypes using Prism 7 software (GraphPad).
5
Immunofluorescence Imaging of Phospho-SMAD3 and YAP-TAZ
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Cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and blocked with 1% bovine serum albumin. Primary antibodies for phospho-SMAD3 (1/500; ab52903; Abcam) and YAP-TAZ (1/1000; 93,622; Cell Signaling Technologies) were incubated one hour. Phalloidin staining was achieved simultaneously (Alexa 594 Phalloidin, A12381, ThermoFisher Scientific) Secondary antibodies used were Alexa 488- donkey anti-rabbit (Jackson Immunoresearch Laboratories, West Grove, PA; 1:500). Nuclear staining was achieved by 20 min incubation at room temperature in Hoechst 33,342 (ThermoFisher Scientific). No cellular autofluorescence and no nonspecific labeling were detected in these conditions. Images were collected by confocal microscopy (Zeiss LSM8) and processed using ZEN (Zeiss) and ImageJ software.
6
Staining F-Actin in MDA-MB-231 Cells
7
Western Blot Analysis of Protein Expression
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Cells were lysed using RIPA lysis buffer [50 mM Tris (pH 7.5), 150 mM NaCl, 1% NP40, 0.25% sodium deoxycholate, 1 mM EDTA, 1 mM DTT and protease inhibitor cocktail]. Protein concentration was determined using the Bradford Protein Assay kit (500–0001; Bio-Rad). Equal amounts of protein were separated on SDS polyacrylamide gels and transferred to nitrocellulose membranes (162–0115; Bio-Rad) using the Bio-Rad semi-dry transfer apparatus. Membranes were blocked for 1 h with 5% skim milk in Tris-buffered saline containing 0.1% Tween-20, and then incubated overnight with primary antibody. Blots were then washed and incubated with secondary antibody, washed again, and visualized by chemiluminescence. β-actin (sc-81178) and α-actinin (sc-166524) were used as loading controls. The TIP60 serum antibody was generated in the lab. E-cadherin (BD-Bioscience), Vimentin (Cell Signaling), Alexa 594 secondary antibody and Alexa 594 phalloidin staining was bought from Life Technologies and mounting medium containing DAPI was purchased from Santa Cruz Biotechnology Inc.
8
Whole Mount Immunofluorescence of Cochlea
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For whole mount immunofluorescence of cochlea, temporal bones of littermate were obtained at 5 days after birth and fixed in 4% paraformaldehyde at 4℃ overnight. The organ of Corti were dissected from temporal bones. Samples were incubated in 5% Triton-X for 30 min and blocked with 1X PBS containing 5% horse serum and 3% BSA for 1 hr. Tissues were stained at 4℃ overnight by anti-myosin7a (1:250, Abcam, Cambridge, Cambridgeshire, UK). Next, the samples were washed in 1X PBS at 10 times for 15 min and incubated overnight at 4℃ with goat-anti-IgG Alexa 488 secondary antibody (1:300, Abcam, Cambridge, Cambridgeshire, UK) and Alexa 594 Phalloidin (1:250, Life Technologies, Carlsbad, California, USA). Finally, the tissues were washed with 1X PBS at 10 times for 15 min and mounted with antifade on glass slides. Pictures of outer hair cells (OHCs) and inner hair cells (IHCs) were taken from parts of the apex, middle, and base of the cochlea duct using confocal microscopy (LSM 710; Carl Zeiss, Oberkochen, Baden-Württemberg, Germany). Then the intensity of primary antibody was measured using a ZEN program.
9
Visualizing Pupal Wing Development
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Fly pupae at white stage were collected and cultured at 29°C for 25 or 28.5 h. Wings were dissected in PBS-t and fixed for 1 h with 4% paraformaldehyde (PFA). Pupae were washed in PBS-t three times for 5 min and blocked in PBS-t with 2% bovine serum albumin (BSA) for 45 min. Dissected pupae were incubated overnight with primary antibody at room temperature in PBS-t-BSA. After incubation, samples were washed five times in PBS-t and incubated in fluorescent phalloidin and fluorescent secondary antibodies for 90 min, both diluted in PBS-t-BSA. Five additional washes were performed in PBS-t, and pupal wings were detached from the pupal cage and mounted on slides with medium for fluorescence (Vectashield, Vector Laboratories). To stain the cellular membrane was used anti-Fmi (from DSHB). Secondary antibody conjugated with the fluorophore Alexa-405 (Invitrogen) and Alexa 594-phalloidin (Invitrogen) were used at 1:200.
10
Immunofluorescence Staining of Epithelial Markers
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Cells grown on coverslips were fixed in 4% paraformaldehyde, permeabilized with 0.2% Triton X-100, blocked with 0.5% BSA/PBS and incubated with the appropriate primary antibody at 4°C overnight. The primary antibodies included CDH1 (E-cadherin, Cell Signaling #4065, 1∶100), β-catenin (Santa Cruz SC-7963, 1∶100) and vimentin (Cell Signaling #5741, 1∶100) antibodies. After washing, the cells were incubated with an Alexa Fluor 594- or 488-lalebed secondary antibody (Invitrogen) for 1 hr, counterstained with DAPI and mounted with SlowFade Gold antifade reagent (Invitrogen). The images were visualized via confocal microscopy using a ZEISS LSM510 META laser-scanning confocal microscope (Carl Zeiss, Germany) with a 63×1.32 NA oil-immersion objective. For WGA staining, cells were incubated with Alexa Fluor555-labeled WGA (Invitrogen, 1∶800) for 10 min at 37°C prior to washing and fixation. For F-actin staining, cells were fixed, permeabilized and incubated with Alexa594-phalloidin (Invitrogen, 1∶1000) for 20 min at 4°C. After washing, the cells were counterstained with DAPI and mounted.
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