Confocal images were acquired using an Olympus Fluoview FV1200 confocal microscope and Olympus FV10-ASW v4.1 software. Olympus UPlanSApo 60X/1.20W and Olympus UPlanSApo 10X/0.45 objectives were used in this study. For all confocal imaging, embryos were embedded on cover slips in 1% low melt agarose. For analysis of filopodia dynamics, z-stacks of the leading edge of NC streams 1–2 (cranial, n = 6) or of trunk NC cells between somites 6–8 (trunk, n = 6) in 26 hpf Tg(sox10:rfpmb) and Tg(sox10:rfpmb); fscn1a MZ embryos were acquired every 4 minutes for one hour using the 60X water objective (S1 –S2 Movies). To monitor NC migration and individual cell behaviors, z-stacks were acquired every 25 minutes for 18 hours using the 10X objective (S3 –S4 Movies). Widefield fluorescent images were acquired on an Olympus SZX16 microscope configured with an Olympus DP72 camera. Brightfield images were taken using a Nikon C-DSD115 microscope configured with an Olympus DP72 camera. Prism 6, ImageJ 1.46r, Adobe Photoshop CS5 and CS6, and Adobe Illustrator CS6 were used to generate figures.
Fv10 asw v4
Manufactured by Olympus
Sourced in Japan
The FV10-ASW v4.1 software is a core imaging software solution for Olympus microscopes. It provides essential functionalities for image acquisition, processing, and analysis. The software enables users to control various microscope settings and capture high-quality images.
Automatically generated - may contain errors
Lab products found in correlation
Dp72 camera, by Olympus (2 mentions)
Szx16 microscope, by Olympus (2 mentions)
Prism 6, by Adobe (2 mentions)
C dsd115 microscope, by Olympus (2 mentions)
Imagej 1.46r, by Adobe (2 mentions)
Fluoview fv1200 confocal microscope, by Olympus (2 mentions)
Fv10i, by Olympus (2 mentions)
Illustrator cs6, by Adobe (1 mentions)
Uplansapo 60x 1.20w, by Olympus (1 mentions)
Fv1200, by Olympus (1 mentions)
Axiovert 200 inverted microscope, by Olympus (1 mentions)
Uplansapo 10x, by Olympus (1 mentions)
Anti mouse igg dylight 594, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Ab53495, by Abcam (1 mentions)
Alexa 488, by Thermo Fisher Scientific (1 mentions)
Axioplan 2, by Zeiss (1 mentions)
Alexa 594, by Thermo Fisher Scientific (1 mentions)
Thermal cycler dice real time system 2, by Takara Bio (1 mentions)
Mascot software, by Matrix Science (1 mentions)
7 protocols using fv10 asw v4
1
Confocal Imaging of Neural Crest Cells
2
Imaging Cranial Neural Crest Dynamics
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Confocal images were acquired using an Olympus Fluoview FV1000XY, FV10i or FV1200 confocal microscopes and Olympus FV10-ASW v4.1 software. All imaging was performed using Olympus UPlanSApo 60X water and Olympus UPlanSApo 10X objectives. Embryos were embedded in 1% low melt agarose on cover slips for all confocal imaging. For analysis of filopodia dynamics, z-stacks of the leading edge of NC stream 3 in 26 hpf Tg(sox10:rfpmb) embryos injected with tp53MO or tp53MO plus fscn1aMO (n = 5 of each) were acquired every 2 minutes for 1 hour using the 60X water objective. For analysis of NC stream depth, z-stacks were acquired of NC stream 3 in 26 hpf Tg(sox10:rfpmb; sox10:h2a-gfp) embryos injected with tp53MO or tp53MO plus fscn1aMO. Cranial NC migration was imaged in 22, 25, 28 and 36 hpf Tg(sox10:GFP) embryos using a Zeiss Axiovert 200 inverted microscope configured with an Olympus DP72 camera. Widefield fluorescent images were acquired on an Olympus SZX16 microscope configured with an Olympus DP72 camera. Brightfield images were taken using a Nikon C-DSD115 microscope configured with an Olympus DP72 camera. Prism 6, ImageJ 1.46r, Adobe Photoshop CC 2014–2015, and Adobe Illustrator CC 2014–2015 were used to generate figures.
3
HCMV Infection Immunofluorescence Assay
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Cells that were grown on chambered cover slips were infected with HCMV strain NR-1 at a multiplicity of infection (MOI) of 5. At 6 h posttransfection, cells were fixed with 4% formaldehyde and blocked with 4% bovine serum albumin (BSA) in PBS and stained with primary mouse IE1/2 antibody (ab53495, Abcam, Cambridge, UK), and then incubated with the secondary antibody Dylight 594 anti-mouse IgG (Life Technology). Cell nucleus was stained with 4′,6-diamidino-2-phenylindole (DAPI) (Invitrogen). Images were captured with a Nikon Eclipse TE300 microscope (Diagnostic Instruments, Inc., Sterling Heights, MI, USA) [20 (link)]. The digital images were subsequently merged using FV10 ASW V4.1 software (Olympus, Tokyo, Japan).
4
Time-Lapse Imaging of Zebrafish Embryos
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Zebrafish embryos were mounted in 35-mm glass-bottom microwell dishes with 1% low-melting-point agarose in E3 egg water. Mounted embryos were submerged in egg water or egg water containing DMSO, TP-0903, RA and/or DEAB. Time-lapse images were acquired using a Olympus Fluoview FV1200 confocal microscope and Olympus FV10-ASW v4.1 software. 10× confocal images were acquired using an Olympus UPlanSApo 10×/0.45 objective every 10-20 min and 60× confocal images were acquired with an UPlanSApo 60×/1.20W objective every 35 min.
5
Immunofluorescence Imaging of Cells
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Cells (5 × 104 cells/mL) on glass coverslips in a 12-well plate were cultured for 24 h and transfected with siRNA or plasmid. After a 48 h incubation, cells were fixed in 4% formaldehyde in PBS, permeabilized with 0.1% Triton X-100 in PBS, and blocked with 6% bovine serum albumin (BSA) in PBS. The coverslips were reacted with primary antibodies in 2% BSA in PBS, secondary antibody conjugated with Alexa-488 or Alexa-594 (Molecular Probes, Eugene, OR) and DAPI to counterstain the nuclei. Fluorescence images were obtained with a fluorescent microscopy, Axioplan 2 (Carl Zeiss, Germany) or FV10i (Olympus, Tokyo, Japan), a laser scanning confocal microscopy, using the ×60 objective lens. Line Plot analysis was performed using FV10-ASW v4.1 software (Olympus).
6
Quantitative analysis of cellular data
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RT-qPCR results were quantified using Thermal Cycler Dice real time system II (TakaraBio). FISH data was quantified using ImageJ software. Immunofluorescence staining data were quantified using FV10-ASW v4.1 software (Olympus). LC-MS/MS data were analyzed using Mascot software (Matrix Science, London, UK). The statistical significance for two-group and multiple comparisons was tested using R software69 , as indicated in the legend of each figure. Non-adjusted (two-group comparison) and adjusted (multiple comparisons) P-values are indicated in each figure. In box plots, the first and third quartiles are indicated by both ends of the box, the median is indicated by a vertical line in the box, and the minimum and maximum excluding outliers are the ends of the whiskers. The outliers are indicated with open circles.
7
Live-Cell Imaging and Nuclear Rupture
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Live cells expressing fluorescently-tagged proteins of interest were seeded onto 35-mm glass-bottom FluoroDishes in DMEM 48 hours before imaging. The day of imaging, the media was removed and replaced with pre-warmed phenol red-free DMEM with Hepes and FBS for imaging (Gibco). Cells were imaged on an Olympus FV1000 confocal microscope and FV10-ASW v4.1 software, with a temperature controlled chamber set at 37°C and 60×/NA 1.42 Plan Apo N oil immersion objective. GFP and mCherry imaging was completed via the 488-nm and 543-nm scanning lasers, respectively. Laser-induced nuclear rupturing was performed by focusing the 405nm excitation laser at 100% power (tornado scan mode) in a small ROI on the nuclear envelope for 6-8 s. Utilizing the SIM-scan feature allowed for simultaneous imaging and laser-induced rupturing. The rupture reporter cGAS-mCherry was used as a secondary rupture reporter in all cells unless otherwise indicated. GFP-LaA, GFP-LaA, and GFP-BAF nuclear photobleaching was performed using the main scanner and a 488-nm laser at 100% power for 10 s or until no noticeable signal was observed in the nucleoplasmic compartment. All images were processed in Photoshop CC 2017 for cropping and brightness/contrast adjustments when applicable. Videos were made by exporting AVIs from the FV10-ASW v4.1 software and importing them into Windows Movie Maker v8.0.7.5.
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