Cells were plated at 8 × 104 cells/mL on 19 mm (VWR International) coverslips at 500 μL. After overnight incubation, the Cenp-E inhibitor was added for various time periods. Cells were fixed with 1% formaldehyde, quenched with glycine, and then permeabilised with PBST (PBS and 0.1% Triton X-100). For microtubule staining the PEM buffer was used. Cells were pre-extracted with 100 mM Pipes, 1 mM MgCl2, 0.1 mM CaCl2, and 0.1% Triton X-100 for 90 seconds, followed by fixation with 4% formaldehyde in PEM buffer for 10 minutes. Cells were then incubated with sheep anti-Bub1 SB1.3 [49 (link)] and with mouse anti-tubulin TAT1 [63 (link)], for 30 minutes, and then washed and incubated with secondary antibodies Cy2-, and Cy3-, antisheep/mouse (Millipore) for 30 minutes. Hoechst 33358 (Sigma) at 1 μg/mL was then added to the cells, followed by mounting onto slides with 90% glycerol and 20 mM Tris-HCl, pH 8.0. Images were taken at room temperature with a restoration microscope (DeltaVision RT; Applied Precision) using a 100x 1.40 NA Plan Apo objective and a filter set (Sedat Quad; Chroma Technology Corp.). Images were captured with a charge-coupled device camera (CoolSNAP HQ; Photometrics) with a z-optical spacing of 0.2 μm. Raw images were then deconvolved with the SoftWorx software (Applied Precision), and these were then processed, and PhotoShop (Adobe) was used to analyse the images.
Deltavision rt
Manufactured by Cytiva
Sourced in United States
The DeltaVision RT is a high-performance microscopy system designed for advanced live-cell imaging. It provides a powerful platform for a wide range of applications, including cell biology, developmental biology, and neuroscience research. The system features a state-of-the-art optical design, high-speed image acquisition, and advanced image processing capabilities, enabling researchers to capture and analyze complex cellular dynamics with exceptional resolution and precision.
Automatically generated - may contain errors
Lab products found in correlation
Softworx software, by Cytiva (5 mentions)
Uplans apo 100x objective lens, by Olympus (3 mentions)
Ix 70 microscope, by Cytiva (3 mentions)
Softworx software, by GE Healthcare (2 mentions)
Softworx, by Cytiva (2 mentions)
Sedat filter set, by Chroma Technology (2 mentions)
Plan apo 100x, by Olympus (2 mentions)
Prolong diamond antifade, by Thermo Fisher Scientific (2 mentions)
Formaldehyde solution, by Merck Group (2 mentions)
Ix71 microscope, by Olympus (2 mentions)
Softworx 6, by Cytiva (2 mentions)
Hoechst 33358, by Merck Group (1 mentions)
Softworx 3, by Cytiva (1 mentions)
Fetal bovine serum (fbs), by Takara Bio (1 mentions)
Gfp booster, by Proteintech (1 mentions)
U plan s apochromat objective, by Olympus (1 mentions)
P erk, by Cell Signaling Technology (1 mentions)
Odyssey infrared imager, by LI COR (1 mentions)
Irdye 800cw, by LI COR (1 mentions)
Odyssey infrared imaging system, by LI COR (1 mentions)
33 protocols using deltavision rt
1
Centromere Protein E Inhibition Assay
2
Quantitative Fluorescence Microscopy of Yeast Cells
3
Live Cell Imaging of Yeast
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Cells were grown in synthetic dropout media and imaged in log phase (OD600 ~ 0.5). For the temperature shift experiment, cells were grown shaking in a 37°C water bath for 30 min before imaging. Live cells were imaged on a DeltaVision RT wide-field deconvolution microscope (Applied Precision). Images were deconvolved using SoftWoRx 3.5.0 software (Applied Precision). Images were further processed in ImageJ, adjusting only min/max light levels for clarity, and using equivalent processing for all images within an experiment.
4
Imaging and Quantifying Early Zygotes
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Collect images of 3−10 fields and examine at least 25 zygotes per cross.
Use a Deltavision RT epifluorescence microscope with an automated stage (Applied Precision, Inc) and an oil immersion objective (Olympus UPlanApo 100
Capture z-stacks at 0.5–1.0 μm intervals using a CCD digital camera (Photometrics CoolSnap HQ). % transmission = 100%, exposure time 0.05–1 s, field 1040
Examine through-focal series spanning a total of 6 μ in all cases.
Remove out-of-focus light using the Softworks deconvolution program (
Repeat experiments 2–4
5
Quantitative Live-Cell Fluorescence Microscopy
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Images of live cells were acquired at room temperature on a Delta Vision RT (Applied Precision) microscope using a 100×/0.35–1.5 Uplan Apo objective and specific band pass filter sets for GFP. The images were collected using a Coolsnap HQ (Photometrics) camera. Image processing was performed using ImageJ (http://rsbweb.nih.gov/ij/ ). Pixel fluorescence intensity of at least 20 fields per sample (see cell number in Figure Legend) was quantified as described in [17] (link), [21] (link) using Knime software (www.knime.org/knime ).
6
Immunofluorescence Imaging of U2OS LacO Cells
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U2OS LacO cells (a gift from S Janicki) were grown in DMEM supplemented with 8% FBS (Clontech), hygromycin (200 µg/ml), pen/strep (50 µg/ml), and L-glutamine (2 mM). Cells were transfected with the indicated constructs for 48 hr using Fugene HD according to the manufacturer's protocol. Asynchronously growing cells were arrested in prometaphase by the addition of nocodazole (830 nM) for 2–3 hr. Cells plated on 12-mm coverslips were fixed (with 3.7% paraformaldehyde, 0.1% Triton X-100, 100 mM Pipes, pH 6.8, 1 mM MgCl2, and 5 mM EGTA) for 5–10 min. Coverslips were washed with PBS and blocked with 3% BSA in PBS for 1 hr, incubated with primary antibodies (GFP-booster [Chromotek], rabbit-anti-BUBR1 [Bethyl] and CREST/anti-centromere antibodies [Cortex Biochem, Inc.]) for 16 hr at 4°C, washed with PBS containing 0.1% Triton X-100, and incubated with secondary antibodies (goat-anti-rabbit Alexa Fluor 568 and goat anti-human Alexa Fluor 647) for an additional hour at room temperature. Coverslips were then washed, incubated with DAPI for 2 min, and mounted using antifade (ProLong; Molecular Probes, Eugene, OR). All images were acquired on a deconvolution system (DeltaVision RT; Applied Precision, part of GE Healthcare) with a 100×/1.40 NA U Plan S Apochromat objective (Olympus, Shinjuku, Tokyo, Japan) using softWoRx software (Applied Precision).
7
Quantitative Western Blot and Microscopy
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Western blots were carried out using the primary antibodies; P-ERK (Cell Signaling), ERK (Cell Signaling), Tubulin (Sigma), UBE3A (Bethyl Laboratories), ELK1 (Epitomics) and p53 (Calbiochem). Proteins were detected using infrared dye-conjugated secondary antibodies (LI-COR Bioscience, IRDye 800CW and IRDye 680LT). The signal was collected with a LI-COR Odyssey Infrared Imager. Data were quantified using Odyssey software (LI-COR Bioscience, Odyssey Infrared Imaging system application software version 3.0.25). For microscopy, cells were grown on coverslips and fixed in 3.7% Paraformaldehyde for 15 minutes. DNA was staining with Hoechst. Images were acquired on a Delta Vision RT (Applied Precision) restoration microscope using a 100x/1.40 Plan Apo objective. The images were collected using a Coolsnap HQ (Photometrics) camera with a Z optical spacing of 0.2 μm. Raw images were then deconvolved using the Softworx software and maximum intensity projections of these deconvolved images are shown in the results.
8
Fluorescent Microscopy Imaging Protocol
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Samples were mounted on microscope slides coated with a thin layer of 1.2% agarose. Images were acquired with a Zeiss Axiovert 200 M or a Zeiss Axiovert 135 microscope coupled to a Sony Cool-Snap HQ cooled CCD camera (Roper Scientific), and using Metamorph imaging software (Universal Imaging). For membrane staining, cells were mounted on slides coated with 1% agarose supplemented with the membrane dye Nile Red (0.1 μg/ml, Molecular Probes) or by mixing 9 μl of cells with 1 μl of Nile Red solution (12.5 mg/ml) before spotting the sample on the agarose slide. Alternatively, cells were mixed with the membrane dye FM5-95 (Invitrogen), at a final concentration of 0.4 μg/ml. Nucleoids were stained by adding DAPI (0.02 μg/ml, Sigma) to the agarose slide. Images were analyzed and prepared for publication with ImageJ (http://rsb.info.nih.gov/ij/ ). For time-lapse microscopy, strain PG718 was grown in CM supplemented with 0.5% xylose at 30°C until cells reached exponential phase, and subsequently mounted onto a thin semisolid matrix made of CM supplemented with 0.5% xylose and 1.5% low-melting point agarose on a microscope slide. Slides were incubated in a temperature-controlled chamber (30°C) on a Deltavision RT automated microscope (Applied Precision). Phase contrast and GFP images were taken every 10 min.
9
Immunofluorescence Staining Protocol
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The cells were fixed with 4% paraformaldehyde for 20 min at room temperature and permeabilised (0.25% Triton X-100 in PBS) for 5 min at room temperature. The cells were blocked (3% goat serum and 0.1% Triton X-100 in PBS) for 30 min and then in primary antibody (diluted in blocking buffer) overnight at 4 °C. After three 5 min washes in PBS, cells were incubated in the secondary antibody for 2 h. Following three further 5 min washes, coverslips were mounted using Vectasheild hard-set mounting compound containing the nuclear DAPI stain (Vector Laboratories, Burlingame, MA, USA). The images were acquired on a DeltaVision RT (Applied Precision, Issauah, WA, USA) restoration microscope using a ×60/1.42 Plan Apo objective and the Sedat filter set (Chroma 89000; Chroma Technology Corp., Rockingham, VT, USA). The images were collected using a CoolSNAP HQ (Photometrics, Tuscon, AZ, USA) camera with a Z optical spacing of 0.5 μm. The images were deconvolved using Softworx Software (GE Healthcare, Issaquah, WA, USA) and maximum intensity projections of images processed using Image J (http://imagej.nih.gov/ij/ ; National Institute of Health, Bethesda, MD, USA).
10
Immunofluorescence Staining of AID Protein
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Cells were attached to polylysine-coated slides, fixed in 4% paraformaldehyde/PBS, permeabilized with 0.15% Triton X-100 for 2 min, and blocked with 1% BSA/PBS. Anti-AID antibody (1:1000 in blocking buffer) and anti-mouse secondary antibody (Alexa Fluor 594, 1:1000 from Molecular Probes) were used to visualize the tagged protein. Three-dimensional data sets were acquired using a cooled CCD camera (CoolSNAP HQ; Photometrics) on a wide-field microscope (DeltaVision RT; Applied Precision) with a 100× NA 1.4 Plan Apochromat lens. The data sets were deconvolved with softWoRx (Applied Precision), exported as TIFF files, and imported into Adobe Photoshop for final presentation.
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