Cells were either grown on poly-l-lysine coated 4-chambered slides (nunc Lab-TekII, www.thermoscientific.com ) or 13 mm2 glass coverslips. To image cells live, the growth medium was replaced with CO2 independent, phenol red free medium containing NucBlue (www.lifetechnologies.com ) to visualize the DNA. To fix cells for immunofluorescence, coverslips were incubated with 4% formaldehyde (37˚C, 5 min) and permeabilised in 0.15% triton in PBS (37˚C, 2 min). Before immunoprobing, cells were blocked with 1% BSA in PBS, then incubated sequentially for 1 h at room temperature with anti-tubulin (B512, 1/2000, Sigma, T5168) or anti-borealin (1/500, in-house), then texas-red conjugated anti-mouse secondary antibodies (1/200, www.vectorlabs.com ) or Cy5-anti-rabbit antbodies (1/1000, www.abcam.com ). All fixed samples were counterstained with DAPI and mounted in 1% propyl gallate/ glycerol mounting medium. Images were acquired using an Olympus inverted microscope fitted with a x 20, or a x 63 (NA 1.4, oil) objective and operated with Deltavision Software (api.gehealthcare.com). High resolution images are presented as 2D projections of 0.3 μm stepped Z-sections and processed for presentation using Adobe Photoshop, adhering to image manipulation regulations.
Deltavision software
Manufactured by GE Healthcare
DeltaVision software is a microscopy imaging solution developed by GE Healthcare. It is designed to enable high-resolution, multi-dimensional data acquisition and analysis. The software provides a user-friendly interface for managing and processing microscopy images.
Automatically generated - may contain errors
Lab products found in correlation
Deltavision microscope, by GE Healthcare (2 mentions)
Deltavision omx, by GE Healthcare (2 mentions)
Inverted microscope, by Olympus (1 mentions)
Nucblue, by Thermo Fisher Scientific (1 mentions)
Anti tubulin b512, by Merck Group (1 mentions)
Nunc lab tek 2, by Thermo Fisher Scientific (1 mentions)
Vectashield mounting medium with dapi, by Vector Laboratories (1 mentions)
Ab15309, by Abcam (1 mentions)
Alexa fluor 488 goat anti mouse secondary antibody, by Thermo Fisher Scientific (1 mentions)
Ab25630, by Abcam (1 mentions)
Anti vinculin antibody, by Santa Cruz Biotechnology (1 mentions)
Plan apo oil objective, by Olympus (1 mentions)
Mouse monoclonal anti gfp antibody, by Roche (1 mentions)
Tev protease, by GenScript (1 mentions)
Texas red, by Vector Laboratories (1 mentions)
Cy5 conjugated secondary antibodies, by Abcam (1 mentions)
Vectashield, by Vector Laboratories (1 mentions)
Deltavision elite, by Olympus (1 mentions)
Eclipse ti, by Nikon (1 mentions)
Slowfade gold mounting media, by Thermo Fisher Scientific (1 mentions)
7 protocols using deltavision software
1
Immunofluorescence Imaging of Microtubules and Chromosomal Proteins
2
Fluorescent Colocalization of GLUT1 and LAMP1
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HeLa cells were grown on coverslips prior to transfection with either Tax-1 or empty expression plasmid or SNX27 or scramble siRNA. 24 hours post-transfection the cells were fixed with 4% paraformaldehyde for 20 minutes at room temperature followed by permeabilization with 0.1% Triton X-100 for 10 minutes at room temperature. Cells were then blocked with 5% bovine serum albumin (BSA) in 1× PBS for 1 hour at room temperature. Cells were then treated with GLUT1 (Abcam, AB15309; 1:100) and LAMP1 (Abcam, AB25630; 1:300) primary antibodies diluted in a 2% BSA 1× PBS solution overnight at room temperature. Alexa Fluor 568 goat anti rabbit and Alexa Fluor 488 goat anti mouse secondary antibodies (Life Technologies; 1:500) were diluted in 2% BSA 1× PBS solution and incubated on the cells for 1 hour at room temperature. Coverslips were mounted onto slides using VECTASHIELD mounting medium with DAPI (Vector Laboratories) per manufacturer’s instructions. Deconvolution images were acquired at room temperature using an inverted DeltaVision microscope (GE) with an oil immersion 60× objective lens. Subsequent colocalization analysis to determine Pearson’s coefficient of correlation and processing of images were performed using the DeltaVision software (GE).
3
Immunofluorescent Staining of Vinculin
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Cells were cultured on glass coverslips with or without poly-L-lysine, then fixed with 4% formaldehyde, permeabilised using 0.15% Triton X-100 in PBS, and blocked with 1% BSA before immunoprobing with anti-vinculin antibodies (catalogue no. 73614, 1:1000, Santa Cruz Biotechnology, 1 h room temperature), and Cy5-secondary anti-rabbit-IgG antibodies (1:1000, AbCam; 1 h room temperature). Samples were counterstained with 20 nM Rhodamine–phalloidin and DAPI, then mounted with Mowiol. Images were acquired using an inverted (Olympus IX71) microscope with 40× NA1.2 oil and 60× NA1.4 oil objectives, DeltaVision software (GE Healthcare) and a Coolsnap HQ2 camera (Photometrics). Maximum projections of deconvolved 0.3-μm z-stacks prepared in Photoshop are presented.
4
Superresolution Imaging of Class IV Neurons
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The larvae were mounted in 50% glycerol in phosphate-buffered saline between a glass slide and a coverslip. The raw images of the class IV neurons were collected by using a DeltaVision OMX superresolution imaging system (GE Healthcare) equipped with a monochrome sCMOS camera (PCO edge) and a 60× Plan Apo oil objective (NA 1.42) (Olympus). The raw images were then reconstructed and deconvolved by using the DeltaVision software (GE Healthcare) to obtain three-dimensional SIM images.
5
Axoneme Immunofluorescence Imaging Protocol
6
Immunofluorescence Staining of Mitotic Spindle
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Cells were cultured on polylysine coated coverslips, fixed with 4% formaldehyde, and permeablized using 0.15% Triton-x-100 in PBS. They were then blocked with 1% BSA in PBS and immunoprobed with primary antibodies for 1 h at room temperature, then incubated with texas-red (1/200, Vector) or Cy5-conjugated secondary antibodies (1/1000, AbCam). Mitotic spindles were revealed with tubulin (B512, 1/2000, Sigma), borealin with a polyclonal rabbit antibody (1/500, in house), and BubR1 with a polyclonal sheep anti- BubR1 antibody (1/1000, gift from Prof. Stephen Taylor, Manchester). Samples were counterstained with DAPI to reveal DNA, and mounted with Vectashield (Vector Laboratories). Images of fixed cells were acquired using an inverted (Olympus IX71, Delta Vision Elite) microscope fitted with 20x (NA 0.85, oil) or 60x (NA1.4, oil) objectives using DeltaVision software (G.E.Healthcare) and a Coolsnap HQ2 camera (Photometrics). For high magnification images two-dimensional projections were created from deconvolved Z-stacks (0.3 μm sections) and then prepared using Adobe Photoshop.
7
Live-cell and Fixed-cell Imaging Protocol
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Live cells and fixed cells were imaged on an inverted microscope (Ti Eclipse; Nikon) at room temperature using a 40× NA 0.6 Plan Fluor air or a 100× NA 1.4 Plan-Apochromat oil objective lens (Nikon). All images were collected with a cooled back-thinned EM charge-coupled device (CCD) camera (iXon+ DU888; Andor Technology) with a 1× or 1.5× optivar using MicroManager 1.4 software. Live cells were imaged in their usual culture media. For cell tracks, a randomly selected region spanning a tiled series of 10 × 10 or 6 × 6 fields of view was imaged every minute for 20 min. For drug perturbation experiments, cells were imaged every 10 s for 10 min before drug addition, and continuing for 30 min after drug addition. For live-cell myosin experiments, cells were imaged every 3 s. Fixed cells were mounted in Vectashield mounting media (Vector Laboratories).
For 3D structured illumination microscopy experiments, fixed cells were mounted in SlowFade Gold mounting media (Invitrogen) and imaged on a DeltaVision OMX (GE Healthcare) microscope using DeltaVision software (GE Healthcare) with a 100× NA 1.4 UPlan-Apochromat oil objective lens (Olympus) using 0.125 µm z slices at room temperature. Images were collected with a cooled back-thinned EM-CCD camera (Evolve; Photometrics). 3D structured illumination reconstructions were created with the softWoRx imaging suite (GE Healthcare).
For 3D structured illumination microscopy experiments, fixed cells were mounted in SlowFade Gold mounting media (Invitrogen) and imaged on a DeltaVision OMX (GE Healthcare) microscope using DeltaVision software (GE Healthcare) with a 100× NA 1.4 UPlan-Apochromat oil objective lens (Olympus) using 0.125 µm z slices at room temperature. Images were collected with a cooled back-thinned EM-CCD camera (Evolve; Photometrics). 3D structured illumination reconstructions were created with the softWoRx imaging suite (GE Healthcare).
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