Following culture, the gels were fixed in 10% formalin for 15 min and washed with phosphate-buffered saline (PBS) 3 times. The cells were permeabilized with 0.1% Triton in PBS and blocked with 5% normal goat serum (NGS) in PBS for 1 h. After blocking, 1000X Phalloidin (Phalloidin-iFluor 594 Reagent, Abcam) was diluted to 1X in 5% NGS and incubated for 1.5 h in the dark. After staining that actin cytoskeleton, the gels were mounted with Antifade Diamond Mount with NucBlue overnight. 10–15 images (0.045 mm2 field) per gel were acquired using a Leica DMi8 inverted fluorescence microscope. A minimum of 50 nuclei for the 2 days incubation or 150 nuclei for the 7 days incubation were counted per independent gel replicate.
Phalloidin ifluor 594 reagent
Manufactured by Abcam
Sourced in United Kingdom, United States
Phalloidin-iFluor 594 Reagent is a fluorescent conjugate used for labeling and visualizing filamentous actin (F-actin) in cells. It binds specifically to F-actin and can be detected using fluorescence microscopy.
Automatically generated - may contain errors
Lab products found in correlation
Triton x 100, by Merck Group (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Bz x800 microscope, by Keyence (2 mentions)
Dmi8 inverted fluorescence microscope, by Leica (1 mentions)
Dapi 4 6 diamidino 2 phenylindole, by Thermo Fisher Scientific (1 mentions)
Snap23, by Abcam (1 mentions)
Rab27b, by Proteintech (1 mentions)
Vamp7, by Novus Biologicals (1 mentions)
Hrp conjugated anti mouse igg, by Cell Signaling Technology (1 mentions)
Hrp conjugated anti rabbit igg, by Cell Signaling Technology (1 mentions)
β actin, by Proteintech (1 mentions)
Rab7 d95f2, by Cell Signaling Technology (1 mentions)
Mrq 13, by Cell Marque (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Axiovert 40 cfl microscope, by Zeiss (1 mentions)
Dmi8 fluorescence microscope, by Leica (1 mentions)
4 6 diamidino 2 phenylindole dapi, by Abcam (1 mentions)
Ab31163, by Abcam (1 mentions)
Alexa fluor 488 goat anti rabbit igg, by Abcam (1 mentions)
26 protocols using phalloidin ifluor 594 reagent
1
Quantifying Actin Cytoskeleton in Cell Cultures
2
Immunofluorescence Evaluation of Osteoclast Differentiation
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On day 14 of OC culture, cells were fixed using paraformaldehyde 4% (Carl Roth, Karlsruhe, Germany). Immunofluorescence staining was performed to evaluate the morphology of OCs and validate the method used for OC differentiation. Therefore, the cells were permeabilized using 0.1% Triton-X 100 (Sigma Aldrich, St. Louis, MO, USA) and 1% bovine serum albumin (BSA; Sigma Aldrich, St. Louis, MO, USA) was used as a blocking reagent. To categorize the cells into different stages of osteoclastogenesis, we used the following reagents: an OC-specific polyclonal anti-human Calcitonin Receptor (CT-R) antibody (Biozol, Eching, Germany) and a recombinant anti-TRAP (Tartrate-resistant acid phosphatase) antibody (Abcam, Cambridge, UK) to stain OC and OC precursors; the Phalloidin-iFluor 594 Reagent (Abcam, Cambridge, UK) for detecting actin filaments in the cytoskeleton; and DAPI (4’,6-Diamidino-2-Phenylindole, Thermo Fisher Scientific, Waltham, MA, USA) for staining cell nuclei. TRAP+ cells with less than three nuclei were considered as OC precursor cells, whereas cells that were TRAP+ CalcR+ with three or more nuclei were defined as mature OCs.
3
Immunoblotting and Immunofluorescence Analysis of Extracellular Vesicle Proteins
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The antibodies used for immunoblotting and immunofluorescence were as follows: Cortactin (H222) (3503; Cell Signaling Technology), VAMP7 (NB100–91356; Novus Biologicals), RAB7 (D95F2) (9367 T; Cell Signaling Technology), RAB27B (13412–1-AP; Proteintech), CD63 (GTX28219; GeneTex), β-actin (20536–1-AP; Proteintech), TSG101(28283–1-AP; Proteintech), CD81(A5270; ABclonal), SNAP23 (ab4114; Abcam), HRP-conjugated anti-rabbit IgG (7074S; Cell Signaling Technology), and HRP-conjugated anti-mouse IgG (7076S; Cell Signaling Technology). The reagent used for immunoblotting was Phalloidin-iFluor 594 reagent (ab176757; Abcam).
4
Fluorescent Staining of CD44 and Hyaluronan
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The cells were cultured on chambered coverglasses (Ibidi GmbH, Martinsried, Germany). For fluorescent stainings, the cells were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS), pH 7.4 for 20 min. The cells were washed with PBS, permeabilized for 20 min at room temperature with 0.1% Triton-X-100 in 1% BSA and incubated overnight at 4 °C with anti-CD44 monoclonal antibody (1:100, MRQ-13, Cell Marque, Rocklin, CA, USA). After washing, the cells were incubated overnight with fluorescently labelled secondary antibody (l:500; Vector Laboratories Inc., Burlingame, CA, USA). For HA staining, monolayer cultures were incubated overnight at 4 °C with 3 µg/mL of biotinylated HA-binding complex (bHABC). After washing, the sections were incubated for 1 h with Texas red-labelled streptavidin (1:500, Vector, Burlingame, CA, USA). Actin filaments were stained with Phalloidin-iFluor 594 Reagent (Abcam, Cambridge, UK). Nuclei were labeled with 4′,6-diamidino-2-phenylindole (DAPI, 1 μg/mL, Sigma-Aldrich, St. Louis, MO, USA).
5
Mesenchymal Stem Cell Immunostaining
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BMMSCs in 24-well plates were fixed in 4% paraformaldehyde for 15 min and permeabilized by 0.1% Triton X-100 (Sigma-Aldrich) for 10 min. The cells were blocked in 1% bovine serum for 30 min and incubated for 1 h in anti-GPx1 primary antibody (1:200). After washing with PBS, the cells were incubated with goat anti-rabbit IgG (H&L) for 1 h. The cytoskeleton F-actin was stained with Phalloidin-iFluor 594 Reagent (Abcam) for 20 min and the nucleus was counterstained using DAPI. Immunofluorescence images were captured using a Zeiss Axiovert 40CFL microscope (Zeiss, Oberkochen, Germany).
6
Fluorescence-based Analysis of Podocyte Cytoskeletal Changes
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Analysis by fluorescence was performed using a Leica confocal microscope DMi8 (Leica Microsystems, Wetzlar, Germany) and images were processed with ImageJ (v. 1.46 r; National Institutes of Health, Bethesda, MD, USA) software. For this, samples were processed as in [59 (link)] and incubated with anti-sirt-1 (1/100). F-actin cytoskeleton distribution was evaluated by phalloidin staining on treated podocytes with HG and Ang II concentrations. Cells were fixed in 4% paraformaldehyde for 15 min at room temperature (RT), washed three times with PBS, permeabilised with 0.1% Triton X-100, washed and incubated with Phalloidin-iFluor 594 Reagent (1/1000, Abcam, Cambridge, UK) for 1 h at RT. Within the last half hour of the incubation period, cells were stained with DAPI. After incubation, cells were washed 3 times in PBS, mounted and observed.
7
Immunofluorescence Assay for Nrf2 Activation
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Raw 264.7 cells were seeded at a density of 3 × 104 cells/well in a 96-well plate. BMDMs were seeded at a density of 1 × 104 cell/well. These cells were infected with oral streptococci as described above. Cells were fixed with 4% paraformaldehyde, followed by permeabilization with 0.2% Triton-X in PBS for 15 min and blocked for 1 h at room temperature with PBS containing 5% bovine serum albumin (Sigma-Aldrich). Cells were stained with primary antibody against Nrf2 (ab31163; Abcam, MA, USA) overnight at 4 °C. Subsequently, cells were incubated with goat anti-rabbit IgG (Alexa Fluor® 488, ab150077; Abcam) in the dark for 1 h at room temperature. Nuclei and cytoskeleton were sequentially counterstained with 4′6-diamidino-2-phenylindole, DAPI (Abcam) and Phalloidin-iFluor 594 reagent (Abcam) respectively. Representative fluorescence images were captured using a DMi8 fluorescence microscope (Leica Microsystems, Wetzlar, Germany).
8
Actin Cytoskeleton Dynamics Visualization
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Actin cytoskeleton alterations were analyzed using Phalloidin-iFluor 594 reagent (Abcam, Cambridge, UK) at day 0, 7, 14 and 21. Cells were fixed and F-actin fibers were stained with Phalloidin for 60 min at room temperature. Nuclei were stained with 10 µg/ml Hoechst 33342 (Invitrogen, Waltham, USA) for 10 min at room temperature. Visualization was done using a Keyence BZ-X800 microscope.
9
Phalloidin-based Cardiomyocyte Actin Staining
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For phalloidin staining, cardiomyocytes were fixed for 20 min in 4% paraformaldehyde (PFA). Cells were blocked and permeabilized for 1 h in TSA Blocking Reagent (FP1012, PerkinElmer) plus 0.1% triton (T9284, Sigma) at room temperature. Then Phalloidin-iFluor 594 Reagent (ab176757, Abcam) was added at 1:1000 dilution and incubated for 90 min. Finally, after wash with PBS, samples were mounted in Mowiol mounting medium (Mowiol 4-88, Glycerol, 200 mM Tris-HCl pH 8.5 and 2.5% 1,4-diazabicyclo-[2,2,2]-octane). Fluorescence images were obtained with a Leica SP8 confocal microscope with HC PL APO 100x/1.4 oil objective. Actin filament dispersion in adult cardiomyocytes were analyzed using Directionality plugin of Fiji.
10
Immunofluorescence Analysis of Phospho-Chk2, LC3A/B, and Actin
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For immunofluorescence analysis, cells were grown on glass coverslips and fixed in a 4% formaldehyde solution for 15 min at room temperature. After rinsing three times with PBS for 5 min each, the cells were blocked in blocking buffer (1XPBS, 5% normal serum, 0.3% Triton X-100) for 60 min at room temperature. After removing blocking solution, the primary antibodies were diluted in antibody dilution buffer (1XPBS, 1% BSA, 0.3% Triton X-100), and incubated on cells overnight at 4 °C. The coverslips were washed with PBS three times and incubated in fluorescent-dye conjugated secondary antibody diluted in antibody dilution buffer for 2 h at room temperature. The cells were washed with PBS three times and counterstained with DAPI. The cells were examined and photographed by immunofluorescence microscopy. The primary antibodies was used in this assay included Phospho-Chk2 (Thr68) (#2197; Cell Signaling, Danvers, MA, USA), LC3A/B (#12741; Cell Signaling, Danvers, MA, USA). Phalloidin-iFluor 594 reagent (ab176757; Abcam, Cambridge, UK) were used for labeling actin filaments
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