Chemicals that were used in this study: Endothelin-1, BQ123, Tetramethylrhodamine B isothiocyanate-conjugated Phalloidin, and Latrunculin B were purchased from Sigma-Aldrich. GSK429286 and Y27632 were obtained from Selleck-chem. C3 was purchased from Cytoskeleton Inc. Phos-tag-conjugated acrylamide was purchased from Wako Chemicals. DAPI was purchased from Invitrogen.
Tetramethyl rhodamine b isothiocyanate conjugated phalloidin
Manufactured by Merck Group
Sourced in United States
Tetramethyl rhodamine B isothiocyanate-conjugated phalloidin is a fluorescent dye used to label and visualize actin filaments in cells. It binds specifically to F-actin, enabling the detection and analysis of the actin cytoskeleton structure.
Automatically generated - may contain errors
Lab products found in correlation
Hoechst 33342 trihydrochloride, by Thermo Fisher Scientific (3 mentions)
Zen imaging software, by Zeiss (3 mentions)
Airyscan, by Zeiss (3 mentions)
Alexa fluor 488 fluorescent dye, by Thermo Fisher Scientific (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Paraformaldehyde (pfa), by Fujifilm (2 mentions)
Bq123, by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Endothelin 1, by Merck Group (1 mentions)
Phos tag conjugated acrylamide, by Fujifilm (1 mentions)
Latrunculin b, by Merck Group (1 mentions)
Y 27632, by Selleck Chemicals (1 mentions)
Hoechst 33258, by Merck Group (1 mentions)
Anti e cadherin, by Proteintech (1 mentions)
Sp5 laser scanning confocal microscope, by Leica (1 mentions)
Anti vimentin, by Proteintech (1 mentions)
Lsm 880 upright, by Zeiss (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Lsm 980, by Zeiss (1 mentions)
Hoechst, by Merck Group (1 mentions)
9 protocols using tetramethyl rhodamine b isothiocyanate conjugated phalloidin
1
Endothelin-1 Signaling Pathway Analysis
2
Immunostaining Cellular Morphology Characterization
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Cells were seeded on glass coverslip or P(CL-co-DLLA) (crosslinked and non-crosslinked) substrates at a density of 1×104 cells/cm2 and incubated for required time periods. The cells were fixed in 4% paraformaldehyde (PFA; Wako Pure Chemical Industries, Tokyo, Japan) and blocked with 1% bovine serum albumin (BSA; Sigma-Aldrich, St. Louis, MO, USA) (in PBS) for 30 min. The IGFBP 5, E-cadherin and vimentin were stained independently with anti-IGFBP5 antibody, anti-E-cadherin and anti-vimentin respectively (Proteintech, Chicago, IL, USA), and the corresponding secondary antibody conjugated with Alexa Fluor® 488 fluorescent dye (Invitrogen, Carlsbad, CA, USA) for 1 h each. F-actin and nuclei were counterstained with tetramethylrhodamine B isothiocyanate-conjugated phalloidin (Sigma-Aldrich, St. Louis, MO, USA) and Hoechst 33258 (Sigma-Aldrich, St. Louis, MO, USA) respectively. The images were taken by Leica SP5 confocal laser scanning microscope (Leica, Wetzlar, Germany).
3
Visualizing Actin Cytoskeleton and Nuclei
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Cells were fixed with 3.7% (w/v) formaldehyde in PBS at room temperature for 5 mins and, then, permeabilized with 0.1% Triton X-100 for further 5 mins at room temperature.
F-actin cytoskeleton was stained with 50 mg/L of tetramethyl rhodamine B isothiocyanate-conjugated phalloidin (Sigma-Aldrich, St. Louis, MO, USA) for 40 mins at room temperature, followed by incubation with 5 mg/L of trihydrochloride Hoechst 33342 (Thermo Fisher Scientific, Eugene, OR, USA) for 10 mins in the dark to stain cells nuclei. Samples were then washed with PBS, mounted and examined using LSM 880 upright confocal laser scanning microscope with Airyscan (Zeiss, Oberkochen, Germany) with a 63× magnification objective. The following settings were employed to excite the dyes and acquire the images: λex 540–545 nm and λex 340 nm; λem 570–573 nm and λem 488 nm for tetramethyl rhodamine B isothiocyanate-conjugated phalloidin and Hoechst, respectively. The obtained images were processed using ZEN imaging software (Zeiss).29 (link)
F-actin cytoskeleton was stained with 50 mg/L of tetramethyl rhodamine B isothiocyanate-conjugated phalloidin (Sigma-Aldrich, St. Louis, MO, USA) for 40 mins at room temperature, followed by incubation with 5 mg/L of trihydrochloride Hoechst 33342 (Thermo Fisher Scientific, Eugene, OR, USA) for 10 mins in the dark to stain cells nuclei. Samples were then washed with PBS, mounted and examined using LSM 880 upright confocal laser scanning microscope with Airyscan (Zeiss, Oberkochen, Germany) with a 63× magnification objective. The following settings were employed to excite the dyes and acquire the images: λex 540–545 nm and λex 340 nm; λem 570–573 nm and λem 488 nm for tetramethyl rhodamine B isothiocyanate-conjugated phalloidin and Hoechst, respectively. The obtained images were processed using ZEN imaging software (Zeiss).29 (link)
4
Cell Adhesion and Signaling Imaging
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Cells were seeded on the fluidic substrates and coverslip glass at 3 × 104 cells/cm2 and incubated for the required periods. The cells were fixed in 4% paraformaldehyde (Fujifilm Wako Pure Chemical Corporation) and blocked with 2% bovine serum albumin (BSA; Sigma-Aldrich) (in PBS) for 1 h. The paxillin, and p-p38/MAPK were stained independently with anti-paxillin antibody and anti-p-p38/MAPK antibody respectively (Santacruz Biotechnology, Dallas, TX, USA), and the corresponding secondary antibody conjugated with Alexa Fluor® 488 fluorescent dye (Invitrogen) for 1.5 h each. F-actin and nuclei were counterstained with tetra-methyl rhodamine B isothiocyanate-conjugated phalloidin (Sigma-Aldrich) and DAPI (Sigma-Aldrich), respectively. The images were taken using a confocal microscope (Zeiss LSM980, Carl Zeiss AG, Oberkochen, Germany) and processed with Zen Blue System software.
5
Endothelial Cell Cytoskeleton Visualization
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EC (105) were seeded onto each well of 24-well plates containing glass coverslips (VWR Internationa, P24G-1.0-10-F) coated with 100 μg of 2% gelatin (Sigma-Aldrich, G9382). They were incubated overnight at 37 °C with 5% CO2 in EC media to form a monolayer. EC monolayers were then fixed with 4% buffered paraformaldehyde (Sigma-Aldrich, 16005) for 20 min at 4 °C, washed three times with PBS and stained with 1 ng/ml tetramethyl rhodamine B isothiocyanate–conjugated phalloidin (Sigma-Aldrich, P1951) for 30 min at 37 °C. Coverslips were extensively washed, air dried, and mounted in Vectorshield (Vector Laboratories, H-5000) mounting medium for fluorescence with DAPI (Vector Laboratories, H-1800) or Hoechst (Sigma, 94403) on glass slides. The slides were analyzed with wide-field fluorescence microscopy.
6
Visualizing F-Actin and Nuclei
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Paraffin sections were treated in xylene and rehydrated in an ethanol and water gradient. To detect F-actin, sections were incubated with tetramethyl rhodamine B isothiocyanate-conjugated phalloidin (0.5 μg/ml, Sigma-Aldrich, USA) for 2 h in the dark [37 (link)]. Sections were then washed and counterstained with DAPI to detect nucleic acid.
7
Immunofluorescence Staining of Endplate Chondrocytes
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Constructs were embedded in O.C.T. prior to sectioning. Subsequently, the sections of cells embedded in agarose were fixed with 4% paraformaldehyde and permeabilized with 0.25% Triton X-100 for 20 min. The whole sections were then blocked with 3% BSA at room temperature for 1 h; then sections were incubated overnight with a rabbit monoclonal antibody (Abcam) recognizing rat Ank, at a dilution of 1:100. Incubation with goat anti-rat fluorescein secondary antibody for 40 min. DAPI is a DNA-binding dye used for nucleus staining. The F-actin cytoskeleton in endplate chondrocytes was stained with tetramethylrhodamine B isothiocyanate-conjugated phalloidin 1:500 (Sigma-Aldrich, St. Louis, MO, USA). Cells were visualized with a confocal microscope (LEICA TCSSP5, Wetzlar, Germany).
8
Visualizing Saos-2 Cells on Bone Cement
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Saos-2 cells were cultured on the bone cement samples for 4 days; after this time cells were washed thoroughly three times in PBS. Cells were then fixed with 3.7% (w/v) formaldehyde in PBS at room temperature for 5 min and permeabilised with 0.1%Triton X-100 at room temperature for 5 min. Then cells were stained with 50 mg/L of tetramethyl rhodamine B isothiocyanate-conjugated phalloidin (Sigma-Aldrich, St. Louis, MO, USA) for 40 minutes at room temperature, followed by incubation with 5 mg/l of trihydrochloride Hoechst 33342 (Thermo Fisher Scientific, Eugene, OR, USA) for 10 minutes in the dark. After washing with PBS, samples were mounted and examined using LSM 880 upright confocal laser scanning microscope with Airyscan (Zeiss, Oberkochen, Germany) for visualization of the staining with a 63X magnification objective. Processing of the obtained images was conducted using ZEN imaging software (Zeiss).
9
Visualizing Metal Nanoparticle Effects on Saos-2 Cells
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Saos-2 cells were cultured and exposed to the various concentrations of metal nanoparticles as stated above in 24-well plates; after the required exposure time cells were at first washed thoroughly three times in PBS. For the staining of the F-actin cytoskeleton and nuclei, cells were fixed with 3.7% (w/v) formaldehyde in PBS at room Temperature for 5 min and permeabilised with 0.1%Triton X-100 at room Temperature for 5 min. Then cells were stained with 50mg/L of tetramethyl rhodamine B isothiocyanate-conjugated phalloidin (Sigma-Aldrich, St. Louis, MO, USA) for 40 minutes at room temperature, followed by incubation with 5mg/L of trihydrochloride Hoechst 33342 (Thermo Fisher Scientific, Eugene, OR, USA) for 10 minutes in the dark. After washing with PBS, samples were mounted and examined using LSM 880 upright confocal laser scanning microscope with Airyscan (Zeiss, Oberkochen,
Germany) for visualization of the staining with a 63X magnification objective.
Processing of the obtained images was conducted using ZEN imaging software (Zeiss). λex 540-545 nm and λex 340 nm; λem 570-573 nm and λem 488 nm for tetramethyl rhodamine B isothiocyanate-conjugated phalloidin and Hoechst, respectively.
Germany) for visualization of the staining with a 63X magnification objective.
Processing of the obtained images was conducted using ZEN imaging software (Zeiss). λex 540-545 nm and λex 340 nm; λem 570-573 nm and λem 488 nm for tetramethyl rhodamine B isothiocyanate-conjugated phalloidin and Hoechst, respectively.
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