Cells were cultured in 12-well plates on 18 mm coverslips and co-transfected with pHERC5 (or pHERC5-C994A or empty vector), pUbcH8, pUbe1L, pISG15 and pGag (10:5:5.5:1 ratio respectively). Twenty-four hours post-transfection, the coverslips containing the cells were washed twice with PF buffer (1× PBS + 1% FBS), fixed for 10 minutes in 1× PBS containing 5% formaldehyde and 2% sucrose, permeabilized in 1× PBS containing 5% NP-40 and then washed twice more with PF buffer. The coverslips were incubated with primary antibodies for one hour, washed 6× with PF buffer, incubated with secondary antibodies (Alexa Fluor 546 anti-mouse or AlexaFluor 488 anti-rabbit, Invitrogen) for one hour and then washed 6× with PF buffer. Coverslips were mounted onto glass slides with ~10 μl of Vectashield mounting media with DAPI (Vector Laboratories) and then sealed with nail polish. Slides were examined using a Zeiss LSM 510 confocal fluorescence microscope and images were obtained with sequential imaging.
Lsm 510 confocal fluorescence microscope
Manufactured by Zeiss
Sourced in Germany
The LSM 510 is a confocal fluorescence microscope manufactured by Zeiss. It is designed to capture high-resolution images of fluorescently labeled samples. The microscope uses a laser as the excitation source and a pinhole aperture to achieve optical sectioning, allowing for the acquisition of detailed, three-dimensional images.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 488 anti rabbit, by Thermo Fisher Scientific (2 mentions)
Anti mouse alexa fluor 546, by Thermo Fisher Scientific (1 mentions)
Vectashield mounting media with dapi, by Vector Laboratories (1 mentions)
Cy3 conjugated anti mouse igg, by Abcam (1 mentions)
Mouse anti gfp, by Cell Signaling Technology (1 mentions)
Nucleofector kit 5, by Lonza (1 mentions)
Normal goat serum (ngs), by Merck Group (1 mentions)
Alexa fluor 555 conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (1 mentions)
Dapi 4 6 diamidino 2 phenylindole, by Thermo Fisher Scientific (1 mentions)
Glass bottom culture dishes, by MatTek (1 mentions)
Alexa fluor 594 anti mouse, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 546, by Abcam (1 mentions)
Anti ve cadherin, by Cell Signaling Technology (1 mentions)
Alexa fluor 488, by Abcam (1 mentions)
Fluorescent mounting medium, by Agilent Technologies (1 mentions)
8 protocols using lsm 510 confocal fluorescence microscope
1
Visualizing HERC5-mediated ISG15 conjugation
2
Immunofluorescence Analysis of VEGFR-3 and VE-cadherin
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The HUtMECs were cultured on glass slides, fixed with cold acetone, and blocked with 5% donkey serum in Tris-buffered saline, 0.1% Tween-20 (TBST). The cells were incubated with goat polyclonal anti-VEGFR-3 (goat polyclonal; cat. no. AF743; R&D Systems) and mouse monoclonal anti-VE-cadherin (mouse monoclonal; cat. no. sc-9989; Santa Cruz Biotechnology, Inc.) at 4°C, followed by treatment with FITC-conjugated anti-goat IgG (cat. no. ab6881; Abcam) and Cy3-conjugated anti-mouse IgG (cat. no. 715-165-150; Jackson ImmunoResearch Laboratories). Nuclei were stained with DAPI (cat. no. BML-AP402; Enzo Life Sciences, Inc.). Samples were mounted in a fluorescent mounting medium and immunofluorescent images were acquired using a Zeiss LSM510 confocal fluorescence microscope.
3
Visualizing F-actin Remodeling in RBL-2H3 Cells
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RBL-2H3 cells were attached and spread on the glass coverslips in 12-well tissue cultured plates, treated with 1 ml cell culture medium containing 1 μg/ml IgE overnight. After being washed with PBS 3 times, the cells were stimulated with 100 ng/ml DNP-BSA for the indicated time and then fixed with 4% paraformaldehyde for 30 minutes. PBS containing 0.5% Triton X-100 was used to permeabilize cell membrane for 30 minutes. Permeabilized cells were blocked with 5% BSA for 1 hour. Then the cells were stained with phalloidin (Beyotime) for 1 hour at room temperature, washed three times with PBS, counterstained with DAPI for 5 minutes. Photographs of stained cells were captured by the LSM 510 confocal fluorescence microscope (Zeiss).
4
Visualizing NR4A1 Localization in HepG2 Cells
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Approximately 1.2 × 106 HepG2 cells were transfected with 4 μg of human NR4A1 plasmid DNA per electroporation using the Nucleofector Kit V (Amaxa, Gaithersburg, MD, USA), according to the manufacturer’s instructions, and cultured for 30 h. The transfected cells were seeded in 8-well chamber slides. Following a 30-h growth period, the transfected cells were fasted in DMEM with 1% FBS for 16 h. The cells were treated for 2 h with a recombinant fusion protein (20 μg/mL) consisting of human ApoA-IV (r-h-apoA-IV) and green fluorescent protein (GFP), which had been purified as described previously [23 (link)]. The cells were fixed in the chamber slides using 4% paraformaldehyde, and permeabilized with 0.2% Triton X-100. Non-specific binding was blocked with 5% normal goat serum (Sigma-Aldrich, St. Louis, MO, USA). The cells were incubated with 1:200 rabbit anti-human NR4A1 (Santa Cruz Biotechnology, Dallas, TX, USA) and 1:200 mouse anti-GFP (Cell Signaling Technology) primary antibodies overnight at 4°C, followed by incubation with 1:200 Alex Flour-594-conjugated goat anti-rabbit (Invitrogen) and 1:200 fluorescein-isothiocyanate (FITC)-conjugated goat anti-mouse secondary antibodies (Invitrogen). The cells were viewed using a Zeiss LSM-510 confocal fluorescence microscope.
5
Immunofluorescent Staining of Cardiac Fibroblasts
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Cardiac fibroblasts were cultured in 35 mm FluoroDish cell culture dishes. The cells were rinsed with PBS, fixed for 20 min at room temperature using PBS containing 2% (v/v) formaldehyde, and washed again with PBS. After permeabilization and blocking with PBS containing 0.2% (v/v) Triton X-100 and 2% (v/v) normal donkey serum, cells were incubated overnight at 4°C with primary antibodies (1:100). The next morning, cells were washed three times with PBS and incubated in the dark with the appropriate Alexa Fluor 555-conjugated secondary antibody (1:500, Life technologies) and DAPI (1:1000) for 1 h at room temperature, rinsed three times with PBS, and mounted on glass slides using 15 μl of DABCO/glycerol. Fluorescence images were acquired using a Zeiss LSM-510 confocal fluorescence microscope. Confocal z-stacks were deconvolved (Huygens Professional, Scientific Volume Imaging) using experimentally derived point-spread functions and 3-dimensional images rendered using Volocity Visualization software (Quorum Technologies Inc).
6
Immunofluorescence Staining of Cells
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Cells were seeded into a 24-well culture plate with glass coverslips, then treated with IFN-γ/LPS for the indicated time. Following washing with phosphate-buffered saline (PBS), cells were fixed in 4% paraformaldehyde for 20 min at room temperature. Fixed cells were washed with PBS for three times and then permeabilized with 0.1% Triton X-100 (v/v) for 20 min. Permeabilized cells were washed with PBS and then blocked with 1% bovine serum albumin for 1 h at room temperature. The cells were then stained with F-actin and α-tubulin antibodies (Beyotime, Shanghai, China) overnight at 4 °C, washed three times with PBS, and counterstained with DAPI for 5 min. Stained cells were photographed with a Zeiss LSM 510 confocal fluorescence microscope (Zeiss, Oberkochen, Germany).
7
Immunofluorescence Staining of Fibroblasts
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Fibroblasts were grown on glass bottom culture dishes (MatTek Corporation, Ashland, MA, USA), washed with PBS and fixed in 2% paraformaldehyde for 30 min at room temperature (RT). Fixed cells were permeabilized with PBS + 0.1% Triton for 10 min at RT and then blocked using PBS + 10% FBS for 30 min at RT. The cells were then incubated with the following primary antibodies in PBS + 5% FBS for 1 h at RT: LRPPRC (1/500), pyruvate dehydrogenase (PDH; Abcam, 1/2000). After three 5 min washes with PBS + 5% FBS, cells were incubated with the following secondary antibodies in PBS + 5% FBS for 1 h at RT: anti-rabbit AlexaFluor 488 and anti-mouse AlexaFluor 594 (Invitrogen, 1/1000). Cells were then washed three times (5 min each) in PBS. Slides were mounted with Prolong Gold antifade reagent containing the nucleus specific staining DAPI (4',6-Diamidino-2-Phenylindole, Invitrogen). Immunocytofluorescence was viewed on a Zeiss LSM 510 confocal fluorescence microscope (Carl Zeiss, Oberkochen, Germany) and image analysis was performed using Zen 2009 Light Edition software.
8
Immunofluorescent Imaging of Angiogenic Markers
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HUtMEC were cultured on glass slides, fixed with cold acetone, and blocked by 5% FBS in PBST. They were incubated with anti-Ang-2 (rabbit polyclonal; proteintech TM; cat. no. 24613-1-AP) and anti-VE-cadherin (rabbit polyclonal, Cell Signaling; cat. no. 2500) at 4°C. Cells were incubated with anti-rabbit IgG conjugated with Alexa Fluor®546 (Abcam; cat. no. ab60317) and anti-human Ig G conjugated with Alexa Fluor®488 (Abcam; cat. no. ab150129). Nuclei were stained with 4′,6-diamidino-2-phenylindole phenylindole (Enzo; cat. no. BML-AP402). Afterward, the samples were mounted in fluorescent mounting medium (DAKO; cat. no. S3023) and immunofluorescent images were acquired using a Zeiss LSM510 confocal fluorescence microscope (Carl Zeiss).
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