Cells were fixed with 4% paraformaldehyde in PBS for 15 min at room temperature, and permeabilized with 0.5% Triton X-100 in PBS for 15 min. The cells were blocked with 1% bovine serum albumin in PBS for 30 minutes and then incubated with rhodamine phalloidin probe (Invitrogen) for 1 h at room temperature. After 3 washes in PBS, the cells were stained with DAPI at room temperature for 10 minutes. The cells were washed 3 times with PBS and mounted with FluorSave Reagent (Calbiochem). The slides were analyzed by an image analysis system (Eclipse E600, Nikon).
Rhodamine phalloidin probe
Manufactured by Thermo Fisher Scientific
Rhodamine phalloidin probe is a fluorescent dye that binds to F-actin, a component of the cytoskeleton in eukaryotic cells. It is commonly used in microscopy and flow cytometry applications to visualize and quantify actin filaments.
Automatically generated - may contain errors
Lab products found in correlation
Axio imager, by Zeiss (2 mentions)
Eclipse e600, by Nikon (1 mentions)
Tritontm x 100, by Merck Group (1 mentions)
Bz x710, by Keyence (1 mentions)
Prolong gold antifade with dapi, by Thermo Fisher Scientific (1 mentions)
Anti grhl2 hpa004820, by Merck Group (1 mentions)
Anti n cadherin, by Takara Bio (1 mentions)
Anti e cadherin, by BD (1 mentions)
4 protocols using rhodamine phalloidin probe
1
Fluorescent Actin Cytoskeleton Imaging
2
Quantifying Residual Hair Cells
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The cultured explants were fixed for 20 min with 4% paraformaldehyde in PBS and permeabilized with 5% TritonTM X-100 (Sigma, St. Louis, MO) in PBS with 10% FBS for 10 min. The specimens were stained with a rhodamine-phalloidin probe (1:100; Invitrogen, Carlsbad, CA) at 15-25°C for 1 h. Phalloidin binds F-actin with nanomolar affinity and labels stereociliary arrays and cuticular plates of hair cells. The specimens were observed under a fluorescence microscope (BZ-X710; Keyence, Osaka, Japan). If no stereocilia or cuticular plate were observed with phalloidin staining, the hair cells were considered missing. Quantitative results were obtained by evaluating 30 outer hair cells associated with 10 inner hair cells [20 (link)]. The average of three separate counts was representative of each culture. The residue of hair cells was measured as a percentage.
3
Visualizing F-Actin and Nuclear Morphology
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NPCs were grown on polyornithine/laminin coated coverslips. Cells were fixed in 4% formaldehyde for 10 min at room temperature and permeabilized with 0.1% Triton X-100 in PBS for 5 min at room temperature. Coverslips were incubated with a rhodamine phalloidin probe (Life Technologies) at a 1:40 dilution for 1 hour at room temperature. Samples were then mounted with ProLong Gold Antifade with DAPI (Life Technologies). Images were taken on a Zeiss AxioImager at 20x magnification. Images were analyzed for F-actin intensity and nuclear size (area) with Volocity (v 6.3). Cell size (area) was analyzed with ImageJ.
4
Immunostaining of Epithelial-Mesenchymal Transition Markers
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Cells grown on glass coverslips were fixed with 4% paraformaldehyde for 10 minutes, and treated with 0.05% Triton-X for 5 min. For immunostaining, fixed cells were incubated with blocking buffer (3% BSA in 1× PBS) for 1 h and then stained with primary antibodies for 1 h at 37 °C. Antibodies used included: anti-GRHL2 (HPA004820, Sigma-Aldrich); anti-E-cadherin (610182, BD); anti-N-cadherin (M142, Takara); anti-pan-Cytokeratin (M3515) and anti-Vimentin (M7020) from Dako; anti-β-Catenin (8480), anti-phospho-Myosin Light Chain 2 (Ser19) (3671), anti-LC3A (4599), anti-EEA1 (3288), anti-LAMP1 (9091), anti-RCAS1 (12290) from Cell Signaling Technology. Alexa Fluor 488-conjugated anti-mouse/rabbit (A11029/A11034) and Alexa Fluor 594-conjugated anti-mouse/rabbit (A11032/A11037) from Invitrogen were used as secondary antibodies. Rhodamine phalloidin probe (R415, Life Technologies) was used for F-actin staining. Cover slips were mounted onto glass slides using Vectashield mounting medium with/without DAPI (H-1200, H-1000) from Vector Laboratories. Images were taken using a Nikon A1R confocal system or a Zeiss AxioImager M2 epifluorescence imaging system.
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