Cells were grown on 3-well chamber microscopy glass slides (Cat. #: 80381, Ibidi, Fitchburg, WI, USA). Slides were fixed with 4% paraformaldehyde (Thermo Fisher Scientific, USA) for 10 min at room temperature. In order to prevent nonspecific background staining, fixed cells were incubated in 1% bovine serum albumin for 30 min. BODIPY (493/503, Thermo Fisher Scientific, USA) and BODIPY (558/568) phalloidin (Thermo Fisher Scientific, USA) diluted in dimethyl sulfoxide (Sigma-Aldrich, USA) at concentration of 1 mg/mL was used to stain neutral fat and F-actin respectively. Cells were then rinsed three times using PBS for 5 min at room temperature. In order to stain for nuclei, cells were stained with 4′,6-diamidino-2-phenylindole (Thermo Fisher Scientific, USA) for 5 min and then washed twice with PBS. The slides were imaged at a magnification of 200× using an inverted fluorescence microscope (Nikon Eclipse, Ti-E; Nikon Instruments Inc., USA) equipped with a UV light source (Nikon Intensilight Inc., USA). All images were analyzed by the NIS Elements imaging software.
Bodipy 558 568 phalloidin
Manufactured by Thermo Fisher Scientific
Sourced in United States
BODIPY (558/568) phalloidin is a fluorescent probe that binds to actin filaments (F-actin) in cells. It is used to visualize the distribution and organization of the actin cytoskeleton in fixed and permeabilized cells.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (3 mentions)
Paraformaldehyde (pfa), by Thermo Fisher Scientific (2 mentions)
Eclipse ti e, by Nikon (2 mentions)
Bodipy 493 503, by Thermo Fisher Scientific (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Horse serum, by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Thermo Fisher Scientific (1 mentions)
Hoecsht 33342, by Thermo Fisher Scientific (1 mentions)
Dmi4000, by Leica (1 mentions)
Mouse anti occludin, by Thermo Fisher Scientific (1 mentions)
Alexfluor 488 secondary antibodies, by Thermo Fisher Scientific (1 mentions)
Tcs spe dmi 4000b, by Leica (1 mentions)
Anti zo 1, by Thermo Fisher Scientific (1 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (1 mentions)
4 protocols using bodipy 558 568 phalloidin
1
Fluorescent Imaging of Cellular Lipids and Cytoskeleton
2
Immunofluorescence Analysis of PPARγ in Differentiated Cells
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Cells were grown on 3-well microscopy glass slides (Ibidi USA, Fitchburg, WI,
USA) for 96 h after inducing differentiation. Slides were fixed with cold 4 %
paraformaldehyde (Thermo Fisher Scientific) for 10 min at room temperature. In
order to prevent nonspecific background staining, fixed cells were incubated in
the blocking solution containing 2% bovine serum albumin (Thermo Fisher
Scientific), 5% of horse serum (Thermo Fisher Scientific), and 0.2% Triton X-100
in PBS for 30 min at room temperature. Fixed cells were then incubated with
primary antibody anti-PPARγ (rabbit polyclonal, dilution 1:100; Abcam,
Cambridge, UK) at 4°C overnight.
The colorization of F-actin was conducted using Bodipy (558/568) phalloidin
(Thermo Fisher Scientific) according to the manufacture’s guidance.
Nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI, Thermo
Fisher Scientific) for 5 min. Slides were imaged at a magnification of 20X using
an inverted fluorescence microscope (Nikon Eclipse, Ti-E; Nikon Instruments)
equipped with a UV light source (C-HGFIE, Nikon Intensilight, Tokyo, Japan). The
NIS Elements® imaging software analyzed all images.
USA) for 96 h after inducing differentiation. Slides were fixed with cold 4 %
paraformaldehyde (Thermo Fisher Scientific) for 10 min at room temperature. In
order to prevent nonspecific background staining, fixed cells were incubated in
the blocking solution containing 2% bovine serum albumin (Thermo Fisher
Scientific), 5% of horse serum (Thermo Fisher Scientific), and 0.2% Triton X-100
in PBS for 30 min at room temperature. Fixed cells were then incubated with
primary antibody anti-PPARγ (rabbit polyclonal, dilution 1:100; Abcam,
Cambridge, UK) at 4°C overnight.
The colorization of F-actin was conducted using Bodipy (558/568) phalloidin
(Thermo Fisher Scientific) according to the manufacture’s guidance.
Nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI, Thermo
Fisher Scientific) for 5 min. Slides were imaged at a magnification of 20X using
an inverted fluorescence microscope (Nikon Eclipse, Ti-E; Nikon Instruments)
equipped with a UV light source (C-HGFIE, Nikon Intensilight, Tokyo, Japan). The
NIS Elements® imaging software analyzed all images.
3
Visualizing Actin and Nuclei in HeLa Cells
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HeLa cells were seeded on the coverslips in 6-well plates with ~2 × 105 cells. Following infection by EDL933-GFP, the wells were washed three times with PBS and then fixed with 3.7% paraformaldehyde at room temperature for 0.5 h. The wells were again washed with PBS twice for 2 min. The samples were quenched with 0.1 M glycine for 15 min. After washing with PBS twice, the samples were blocked and permeabilized by 1% bovine serum albumin + 0.2% Triton X-100 for 15 min. The coverslips were air-dried and overlaid with Bodipy 558/568 phalloidin (Invitrogen, B3475) for 20 min and DAPI (4′,6-diamidino-2-phenylindole) in PBS for 5 min in the dark at room temperature. The coverslips were washed in PBS and mounted on microscope slides by using Fluorescent Mounting Medium. The samples were ready to observe by immunofluorescence microscopy (Nikon Ti-U).
4
Immunofluorescence Microscopy of Epithelial Cells
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Epithelial cells were cultured on glass coverslips in 24-well plates, infected with clinical EPEC isolates, washed with PBS and fixed using either cold methanol (5 min) or 3.7% paraformaldehyde in PBS (20 min), washed with PBS, then permeabilized with 0.1% Triton X-100 (5 min) at room temperature and incubated overnight in blocking solution (Invitrogen, 000–105). Cells were labeled with anti-ZO-1 (Invitrogen 617300) in a 1:100 dilution, mouse anti-occludin (Invitrogen 33–1500) at a 1:100 dilution at 4°C overnight, or 1 U of BODIPY-558/568 Phalloidin (Invitrogen, B3475) diluted into 50 µL of blocking solution for 1 h. Cells were incubated with AlexFluor-488 secondary antibodies (Invitrogen, A11034 or A11029) at 1:250 in Invitrogen blocking solution for 2 h at room temperature, nuclei were stained with Hoecsht 33342 (Invitrogen, H3570) and coverslips mounted using ProLong Gold Antifade reagent (Invitrogen, P36934). Images were acquired using either a Leica DMI4000 (MetaMorph software) fluorescence microscope, or a Leica TCS SPE DMI 4000B (LAS X software) confocal microscope and processed with ImageJ and Adobe Photoshop 2020.
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