For immunofluorescence experiments, cells (40% confluent) were cultured on glass slides, fixed with 4% paraformaldehyde and then washed with PBS 1X. For vinculin analysis, cells were seeded on glass slides coated with 0.5 μg of rat tail collagen I (Invitrogen) o/n at 4°C and blocked with 1% BSA for 1h30. Vinculin antibody (1/200, Sigma) diluted in PBS containing 1% BSA was used. Secondary antibody tagged with Alexa-488 (1/1000, Life Technology) and/or rhodamin-phalloidin (1/750, Sigma) were then added for 1h. Nuclei were counterstained with Hoescht staining. Immunofluorescence was analyzed using Zeiss-AxioImager or LSM780 Confocal microscope. Vinculin quantification was performed using computational analysis.
Rhodamin phalloidin
Manufactured by Merck Group
Sourced in United States, Germany, France
Rhodamin-Phalloidin is a fluorescent dye used for labeling and visualizing actin filaments in cells. It binds specifically to filamentous actin (F-actin), allowing for the detection and analysis of the cytoskeleton structure.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 780 confocal microscope, by Zeiss (3 mentions)
Axio imager, by Zeiss (1 mentions)
Rat tail collagen 1, by Thermo Fisher Scientific (1 mentions)
Alexa 488, by Thermo Fisher Scientific (1 mentions)
Vinculin antibody, by Merck Group (1 mentions)
Fluorescence mounting medium, by Agilent Technologies (1 mentions)
Tcs sp8, by Leica (1 mentions)
Keyhole limpet hemocyanin (klh), by GenScript (1 mentions)
Tcs sp1 laser scanning confocal microscope, by Leica (1 mentions)
Alexa fluor 488 conjugated goat anti rabbit antibody, by Thermo Fisher Scientific (1 mentions)
Fluoview fv1000 confocal microscope, by Olympus (1 mentions)
Alexa 488 goat anti horseradish peroxidase, by Jackson ImmunoResearch (1 mentions)
Nativepage cathode buffer additive, by Thermo Fisher Scientific (1 mentions)
Nativepage running buffer, by Thermo Fisher Scientific (1 mentions)
Fluoromount aqueous mounting medium, by Merck Group (1 mentions)
Fv1000, by Zeiss (1 mentions)
A11001, by Thermo Fisher Scientific (1 mentions)
A11008, by Thermo Fisher Scientific (1 mentions)
A21422, by Thermo Fisher Scientific (1 mentions)
A21428, by Thermo Fisher Scientific (1 mentions)
7 protocols using rhodamin phalloidin
1
Immunofluorescence Analysis of Vinculin
2
Actin Dynamics in Bacterial Internalization
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Jeg-3 and CHO cells on coverslips were infected with either GST-114-159 Rck-, GST-PagN- or GST- coated beads at MOI 50:1. After incubation for 30 min, cells were washed in PBS to remove unbound extracellular beads. In brief, after fixation of the monolayers in PFA 4%, and permeabilization in triton 0.2%, actin was stained with Rhodamin-Phalloidin (diluted 1:200; Sigma;). Finally, coverslips were mounted in fluorescence mounting medium (Dako) and analyzed with a Leica SP8 confocal laser-scanning microscope (Leica TCS SP8, Germany).
3
Localization of Ion Channels in Hydra
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To localize the expression of ANO1-like and SCN5A-like ion channels in Hydra using immunocytochemistry, polyclonal antibodies were raised against synthetic peptides in rabbits. The peptides that correspond to intracellular loops located between transmembrane domains of the ion channels (hySCN5-like: SRSKPKMFKDYKPE; hyANO-like: ETRRPIADRAQD) were synthetized, purified, and N terminally conjugated with KLH prior to injection (GenScript). Polyclonal antibodies were affinity-purified on the antigen and concentrated to 1.5 mg/mL. Serum harvested from the rabbits prior to their immunization was used as control.
Immunohistochemical detection in whole-mount Hydra preparations was carried oud as described previously (77 (link)). Briefly, polyps were relaxed in urethane, fixed in paraformaldehyde, permeabilized with 0.5% Triton X-100 in PBS, incubated in blocking solution for 1 h, and incubated further with primary antibodies diluted to 1.0 μg/mL in blocking solution at 4 °C. AlexaFluor488-conjugated goat anti-rabbit antibodies (Invitrogen) were diluted to 4 μg/mL and incubations were done for 1 h at room temperature. Rhodamin-phalloidin (Sigma) and TO-PRO3-iodide-AlexFluor633 (Invitrogen) counterstaining was conducted as described previously (77 (link)). Confocal laser-scanning microscopy was done using a TCS SP1 laser scanning confocal microscope (Leica).
Immunohistochemical detection in whole-mount Hydra preparations was carried oud as described previously (77 (link)). Briefly, polyps were relaxed in urethane, fixed in paraformaldehyde, permeabilized with 0.5% Triton X-100 in PBS, incubated in blocking solution for 1 h, and incubated further with primary antibodies diluted to 1.0 μg/mL in blocking solution at 4 °C. AlexaFluor488-conjugated goat anti-rabbit antibodies (Invitrogen) were diluted to 4 μg/mL and incubations were done for 1 h at room temperature. Rhodamin-phalloidin (Sigma) and TO-PRO3-iodide-AlexFluor633 (Invitrogen) counterstaining was conducted as described previously (77 (link)). Confocal laser-scanning microscopy was done using a TCS SP1 laser scanning confocal microscope (Leica).
4
Measuring Ovary Size in Drosophila
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Following 7 days on the diets, the female flies were anaesthetized on ice. Ovaries were dissected from 3 females per condition. The ovaries were incubated in 3.7% formaldehyde for 45 min, followed by 3 washes with cold PBS. Fixed ovaries were incubated with rhodamin phalloidin (product #P1951; Sigma-Aldrich, St. Louis, MO, USA) in PBS + 0.4% Triton X-100 (PBST) overnight at 4 °C, followed by 3 washing steps with cold PBST. The ovaries were mounted using Vectashield H1000 fluorescence-mounting medium (Vector Laboratories, Burlingame, CA, USA). Images were taken with an Olympus Fluoview FV1000 confocal microscope (Olympus corporation, Tokyo, Japan) and processed using ImageJ64 (Fiji) [26 (link)]. Using this software, the diameter of the ovaries was measured (in µm).
5
Fluorescent Protein Labeling Protocol
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5-ALA hydrochloride (neo-ALA Co. Ltd, Tokyo, Japan) and SFC (Komatsuya Corporation, Osaka, Japan) were provided by SBI Pharmaceuticals Co., Ltd. Alexa 488 Goat Anti-Horseradish Peroxidase (Jackson Immuno Research), Rhodamin-Phalloidin (Merck/Sigma-Aldrich, Germany), NativePAGE™ Running Buffer and NativePAGE™ Cathode Buffer Additive (Thermo Fisher Scientific, USA) were purchased.
6
Immunofluorescence Staining of Cultured Cells
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Cells were cultured for 24 h on coverslips and then fixed with cold methanol for 20 min at −20 °C. Blocking was realized with 1% BSA for 1 h at 37 °C. Incubations with primary antibodies were performed overnight at 4 °C, and then cells were rinsed 3 times with 0.1% tween-TBS. Incubations with secondary antibodies goat anti-rabbit and goat anti-mouse AlexaFluor 488 or 555 (1/1000, A11008, A21428, A11001, A21422, Life technologies, Carlsbad, CA, USA) were performed for 1 h at 37 °C and cells were then rinsed 3 times with 0.1% tween-TBS, stained with DAPI (4′,6′-diamidino-2-phénylindole) and mounted using Fluoromount Aqueous Mounting Medium (F4680, Sigma Aldrich). ACTIN was stained with Phalloidin-Rhodamin (P1951, Sigma-Aldrich, Saint-Quentin-Fallavier, France). Images were collected with an Olympus FV1000 or Zeiss LSM800 AiryScan laser scanning confocal microscope with a 63X objective.
7
Cell Cycle Synchronization Assay
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Thymidine, nocodazole, phalloidin-rhodamin, propidium iodide and AZD-1152 were from Sigma–Aldrich. RNase A was supplied by Euromedex.
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