Immunofluorescence analysis was performed as described previously (Wang et al., 2016). Briefly, 2 OD cells were fixed by addition of formaldehyde to a final concentration of 3.7% (v/v). Cells were pelleted, resuspended in 500 l of 3.7% formaldehyde in 0.1M potassium phosphate (pH 6.4) and fixed for 15 minutes at room temperature. Cells were washed 3× in in 0.1M potassium phosphate (pH 6.4) and once in sorbitol-citrate (128mM KH2P04, 36mM citrate, 1.2M sorbitol), followed by spheroplasting with zymolyase and glusulase in sorbitol-citrate. Spheroplasts were fixed on polylysine-coated slides, incubated with primary antibody overnight at 4°C, and secondary antibody for 4 hours at room temperature. Antibodies were diluted in PBS/BSA (50mM potassium phosphate, pH 7.4, 150mM NaCl, 1% bovine serum albumin, 0.1% sodium azide). Nop1 was detected using a mouse monoclonal Nop1 antibody (EnCor Biotechnology) at a 1:500 dilution and an anti-mouse fluorescein (FITC) antibody (Jackson ImmunoResearch) at 1:200. Nuclear DNA was detected using DAPI staining (Vectashield). Images were captured as z-stacks using a Delta Vision Elite System with EMCCD camera (Applied Precision, Issaquah, WA). Stacks were deconvolved, and compressed into a 2D image using SoftWoRX2.50 software (Applied Precision).
Deltavision elite system
Manufactured by Cytiva
Sourced in United States
The DeltaVision Elite system is a high-resolution microscopy solution designed for advanced imaging applications. It provides exceptional image quality and advanced features to enable detailed cellular and subcellular analysis.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 488 goat anti mouse, by Thermo Fisher Scientific (2 mentions)
Uplfln 40 1.30na oil immersion objective, by Olympus (2 mentions)
Vectashield, by Vector Laboratories (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Coolsnap hq2 ccd camera, by Teledyne (2 mentions)
Matlab, by MathWorks (2 mentions)
μ slide 8 well glass bottom, by Ibidi (2 mentions)
Axiovert 200m, by Zeiss (2 mentions)
Emccd camera, by Cytiva (1 mentions)
Softworx2, by Cytiva (1 mentions)
Dapi staining, by Vector Laboratories (1 mentions)
Prolong gold, by Thermo Fisher Scientific (1 mentions)
Chamber slide, by Ibidi (1 mentions)
Apo tirf 1.49 na objective, by Nikon (1 mentions)
A1 scanning confocal microscope, by Nikon (1 mentions)
Nis element, by Nikon (1 mentions)
Phalloidin 568, by Thermo Fisher Scientific (1 mentions)
Illustrator software, by Adobe (1 mentions)
Uplsapo oil immersion objective, by Olympus (1 mentions)
Ix71 inverted microscope, by Olympus (1 mentions)
20 protocols using deltavision elite system
1
Immunofluorescence Analysis of Nop1 Localization
2
Immunofluorescence Imaging of von Willebrand Factor
3
Trp53 Gene Localization via DNA-FISH
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Dissociated splenic and thymic cells were fixed using methanol/acetic acid (3:1), washed with fixative three times, and kept in fixative at −20°C until use. To make custom Trp53 BAC probes, the following BACs were ordered from bacpac (https://bacpacresources.org ): RP24-285L20 and RP23-243M15. BACs were isolated from 50-mL bacterial cultures using the BACMAX Bac extraction kit (Epicentre). Isolated BACs were sonicated (10 cycles of 15 sec “on” 45 sec “off” at constant intensity with power set to “3”; Branson Sonifier 450) and labeled with TM-rhodamine Label-IT (Mirus). Labeled BAC probes were suspended in commercial chromosome paint probe for chromosome 11 (Metasystems). DNA-FISH was performed by applying probes onto samples and covering with a glass coverslip. Genomic DNA and probes were codenatured for 2 min at 75°C by placing the slide on a preheated metal plate. Samples were hybridized overnight at 37°C in a dark humidified chamber. Slides were subsequently washed with 0.4× SSC for 2 min at 72°C and rinsed in 2× SSC, 0.05% Tween-20 for 30 sec at room temperature. Slides were then rinsed in PBS, counterstained with DAPI, and mounted using Prolong Gold (Invitrogen). FISH images were acquired on a DeltaVision elite system (Applied Precision) at 40× magnification (10 × 0.5-μm z-sections). Maximum intensity projections were generated using the softWoRx program.
4
Visualizing Centrosomes in Mouse Cells
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Centrosomes were visualized by the centrin-GFP marker in PRG5-derived mouse embryonic fibroblasts grown in chamber slides (ibidi) and were imaged (1 µM, 10×, 40×/1.35 NA) using the DeltaVision elite system (Applied Precision). Centrosomes visualized by the centrin-GFP marker in tissue cryosections were imaged (at 0.2-μm Z-sections with a Nikon 100× APO TIRF 1.49 NA objective) using the Nikon A1 scanning confocal microscope operated with NIS-Elements (Nikon). Additional quantification was done using an Olympus bx43 microscope equipped with a X-Cite series 120Q fluorescence lamp (60× magnification) and using Thermo Scientific Menzel-Gläser Deckgläser Coverslips ø12 mm #1.
5
Immunofluorescence Staining of Cytoskeletal Proteins
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Cells were grown on glass coverslips and fixed with 3.7% formaldehyde in PBS (containing Ca2+ and Mg2+) for 10 min followed by permeabilization for 10 min with 0.1% Triton X-100 in PBS (Ca2+ and Mg2+). Cells were blocked in PBS (Ca2+ and Mg2+) containing 10% FBS and labelled with primary antibodies as indicated. Cells were washed 3 times followed by labelling with Alexa Fluor secondary antibodies. Staining of F-actin was carried out using Phalloidin-568 (Molecular Probes). DNA staining was performed with 4′,6-Diamidino-2-phenylindole (DAPI [Molecular Probes]) after which the coverslips were mounted in Vectashield solution. For endogenous KIF4 and PRC1 stainings, cells were pre-permeabilized for 3–5 min in PHEM buffer (100 mM PIPES pH 6.8, 25 mM HEPES pH 7.4, 5 mM EGTA, 2.5 mM MgCl2) plus 0.5% Triton X-100. Then, cells were fixed for 10 min in PHEM buffer containing 3.7% formaldehyde. Mitotic PRC1 phosphorylation was monitored after ice-cold methanol fixation. Calcium-stable microtubule stainings were carried out as described previously50 (link). Subsequent steps were performed as described above. z-stacks with 0.2 μm spacing were acquired using a DeltaVision Elite system (Applied Precision). Maximum intensity projection of the z-levels were analysed with ImageJ (National Institute of Health) and processed using Photoshop and Illustrator software (Adobe).
6
Immunofluorescence Imaging of Cells
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Cells were grown on glass coverslips and fixed with 3.7% formaldehyde in PBS (containing Ca2+ and Mg2+) for 10 min followed by permeabilisation for 10 min with 0.1% Triton X-100 in PBS (Ca2+ and Mg2+). Cells were blocked in PBS (Ca2+ and Mg2+) containing 10% FBS and labelled with primary antibodies as indicated. Cells were washed 3 times followed by labelling with Alexa Fluor secondary antibodies. DNA staining was performed with 4′,6-Diamidino-2-phenylindole (DAPI [Molecular Probes]) after which the coverslips were mounted in Vectashield solution. Z-stacks with 0.2 µm spacing were acquired using either a Leica AOBS confocal microscope or a DeltaVision Elite system (Applied Precision). Maximum intensity projection of the z-levels were analysed with ImageJ (National Institute of Health) and processed using Photoshop and Illustrator software (Adobe).
7
Widefield Fluorescence Imaging Protocol
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Widefield fluorescence imaging was performed at 20 °C on a DeltaVision Elite system (Applied Precision) equipped with a ×100/1.40 NA UPLSAPO oil immersion objective (Olympus), a CoolSnap HQ2 CCD camera (Photometrics), DAPI (excitation 390/18; emission 435/40) and FITC (excitation 475/28; emission 525/45) filters. 12-bit image stacks were acquired with a z-step of 150 nm giving a voxel size of 64.5 × 64.5 × 150 nm.
8
Correlated Live-Cell Imaging of Cells
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Live-cell imaging and sample examination were performed using a DeltaVision Elite system (Applied Precision, USA), on an Olympus IX71 inverted microscope, running softWoRx 6.0. Fluorescent images were acquired at 10×, 20×, 40× and 60× magnifications, by a CoolSnap HQ2 CCD camera (Roper Scientific, USA). Time-lapse imaging was carried out for 24 hr or 48 hr at 10 min intervals, and acquired at a 20× or 10× magnification (20×/0.5NA or 10×/0.3NA objectives). Correlated live-cell immunofluorescence microscopy was performed on gridded #2 glass-bottomed dishes (MatTek Corporation), using either 10×/0.3NA or the 20×/0.5NA objectives for time-lapse imaging, and the 10×/0.3NA or 20×/0.85NA objectives for immunofluorescence correlation.
9
Visualizing Rab11 Dynamics in Trypanosoma
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Epimastigotes (1×108 cells) in logarithmic phase of growth were collected by centrifugation, washed 3 times in PBS and resuspended in isosmotic buffer (composition mentioned above). GFP-TcRab11 overexpressing epimastigotes were immobilized with poly-L-lysine on coverslips in MatTek glass bottom dishes for 30 min at room temperature. Unattached cells were washed with PBS. To induce hyposmotic stress the isosmotic buffer was diluted by 1∶1 with deionized water. Hyperosmotic stress was induced by bathing the chamber with hyperosmotic buffer (as described above). Time lapse photographic data were collected at 1 sec intervals with a 60× objective and a 1024×1024 field with a Delta Vision Elite system (Applied Precision). Video sequences were reconstructed using Quicktime software.
10
Widefield Fluorescence Imaging Protocol
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Widefield fluorescence imaging was performed at 20°C on a DeltaVision Elite system (Applied Precision) equipped with a 100x/1.40 NA UPLSAPO oil immersion objective (Olympus), a CoolSnap HQ2 CCD camera (Photometrics), DAPI (excitation 390/18; emission 435/40), FITC (excitation 475/28; emission 525/45) and TRITC (excitation 542/27; emission 593/45) filters. 12-bit image stacks were acquired with a z-step of 150 nm giving a voxel size of 64.5 nm x 64.5 nm x 150 nm. Image restoration was carried out using Huygens deconvolution Classic Maximum Likelihood Estimation (Scientific Volume Imaging B.V.).
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