Cells were fixed with 4% PFA, followed by blocking with 1% milk for 30 min. The cells were incubated with mouse anti-HA antibody for 1 h and fixed again in 4% PFA. Cells were then permeabilized with 0.5% Triton X-100 for 5 min and incubated with either rabbit anti-M1 or anti-M2 antibody for 1 h. PLA was carried out using Duolink In Situ PLA kit (Olink Bioscience) according to the manufacturer’s protocol. The mean intensity of the PLA signals was measured using IMARIS software (Carl Zeiss).
Imaris software
Manufactured by Zeiss
Sourced in Germany, Switzerland
Imaris is a powerful 3D and 4D image analysis software developed by Bitplane, a Zurich-based company that is part of the Zeiss group. The software is designed to provide advanced visualization and analysis capabilities for a wide range of microscopy data, including confocal, multiphoton, and light-sheet microscopy. Imaris offers a comprehensive set of tools for segmentation, tracking, and quantification of biological structures in complex 3D and 4D datasets.
Automatically generated - may contain errors
Lab products found in correlation
Duolink in situ pla kit, by Olink (3 mentions)
Lsm 880, by Zeiss (3 mentions)
Stage incubator, by Zeiss (2 mentions)
Csii cmv mcs vector, by RIKEN BioResource Center (2 mentions)
Premiere pro cc, by Adobe (2 mentions)
Osirix md software, by Pixmeo (2 mentions)
Cytospin, by Thermo Fisher Scientific (1 mentions)
Plasmem bright green, by Dojindo Laboratories (1 mentions)
Hepes, by Merck Group (1 mentions)
D5921, by Merck Group (1 mentions)
A1r confocal imaging system, by Nikon (1 mentions)
M5310, by Merck Group (1 mentions)
Lsm 700, by Zeiss (1 mentions)
Calcein am, by Thermo Fisher Scientific (1 mentions)
Phosphate buffered saline (pbs), by Nacalai Tesque (1 mentions)
Metamorph software, by Molecular Devices (1 mentions)
Glass bottom dishes, by MatTek (1 mentions)
Mounting medium, by Vector Laboratories (1 mentions)
Dulbecco s modified eagle medium dmem, by Thermo Fisher Scientific (1 mentions)
Rhodamine b, by Fujifilm (1 mentions)
13 protocols using imaris software
1
Protein-Protein Interaction Analysis by PLA
2
In situ Proximity Ligation Assay for Influenza
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In situ PLA was carried out as previously described48 (link). Infected cells were fixed with 4% PFA for 10 min, followed by permeabilization with PBS containing 0.5% Triton X-100 at room temperature for 5 min. The cells were incubated with PBS containing 1% skim milk for 30 min, and incubated with rabbit anti-M1 and mouse anti-NP antibodies for 1 h. PLA assays were performed using Duolink in situ PLA kit (Olink Bioscience) according to the manufacturer’s protocol. Counting of the PLA foci was carried out using IMARIS software (Carl Zeiss).
3
Visualizing Organelle Dynamics in iNeurons
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HeLa cells and iNeurons (iPSC-derived induced neurons) were transduced with lentivirus encoding VPS29-YFP or baculovirus encoding Rab5a or Rab7a fused with RFP at N-terminal (Life Technologies CellLight Reagents BacMam 2.0; C10587 and C10589, respectively). Lentivirus expression plasmids were constructed by inserting the VPS29-YFP fragment into the CSII-CMV-MCS vector from RIKEN BioResource Research Center. VPS29 and YFP were conjugated by PCR. Lentiviral vectors were generated according to a lentiviral vector preparation protocol from RIKEN BioResource Research Center. Sixteen to twenty-four hours after transduction, cells were imaged at 37 °C in a stage incubator (Carl Zeiss), and time-lapse fluorescence images were acquired with a confocal laser microscope (LSM880, Carl Zeiss). Cells with long neurites were chosen as iNeurons for the experiments. Images of vesicles were captured, and data acquisition was performed using Imaris software (Carl Zeiss). Baculovirus (Life Technologies CellLight Reagents BacMam 2.0; C10597) transduction was used to visualize markers of lysosomes.
4
Fluorescent Labeling and Imaging of Cells
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Cells (1 × 106) were treated with TRITC-FA-HP-β-CyD (1 mM), TRITC-HP-β-CyD (10 mM), and TRITC-FA-HP-β-CyD (1 mM) with folate (4 mM) at 37 °C for 1 h. The medium was removed and PlasMem Bright Green (×200, 200 uL, Dojindo Molecular Technologies) was both added and incubated at 37°C for 5 min. After centrifugation onto glass slides using Cytospin™ (Thermo Fisher Scientific, Waltham, MA, USA), cells were washed gently with PBS containing 0.1% Tween 20 and incubated in PBS (pH 7.4) containing 4% paraformaldehyde for 10 min at room temperature. Then, the samples were incubated with PBS containing 0.1% Triton X-100 for 10 min and washed three times with PBS. To block the non-specific binding of antibodies, the samples were incubated with blocking solution for 30 min at room temperature and slides were sealed with mounting medium with DAPI. For cell observation, the confocal laser microscope LSM-880 (Carl Zeiss, Jena, German) was used. The results were analyzed by Imaris software (Carl Zeiss).
5
Visualizing SARS-CoV-2 Spike Protein Interactions
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Cells were infected with SARS-CoV-2 at an MOI of 100 on ice for 30 min. After the virus adsorption, the cells were washed twice with ice-cold DMEM and then further incubated at 37°C for 5, 10, and 15 min. The cells were then fixed with 4% PFA and incubated with 1% milk for 30 min. The cells were incubated with 10 ng/μl mouse anti-Spike antibody for 1 h and fixed again with 4% PFA. PLA was carried out using a Duolink In Situ PLA kit (Olink Bioscience) with anti-mouse PLUS (DUO92001) and anti-mouse MINUS probes (DUO92004) according to the manufacturer’s protocol. Briefly, the cells were incubated with anti-mouse PLUS and anti-mouse MINUS probes for 1.5 h at room temperature, and then washed with Wash Buffer A. The cells were further incubated with PLA ligase for 45 min at 37°C. After washing, the cells were subjected to the amplification reaction using the polymerase, which is supplied in the kit, for 2.5 h at 37°C. After final washing using Wash Buffer B, the cells were stained with 4,6-diamidino-2-phenylindole (DAPI) for 10 min. The number of PLA signals was measured using IMARIS software (Carl Zeiss).
6
Visualizing PAK, AKT, and Rac1 Dynamics
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MDCK PAK-PBD-AcGFP and MDCK AKT-PH-GFP on a glass bottom dish covered with a collagen gel cultured with media without phenol red (D5921; Sigma) containing 25 mM HEPES (Sigma). To cut down on evaporation of media, we dropped mineral oil (M5310; Sigma) on it. Fluorescent images and transmitted light images were obtained using an A1R confocal imaging system (Nikon) every 5 min. Inhibitors mixed with DMEM were added by micropipette and 1 min after inhibitor treatment, we resumed capturing images. Fluorescent intensity was analyzed using Image J software. GFP-positive cells were selected, and the front-rear signal intensity was calculated using line-plotted intensity data for analysis of MDCK PAK-PBD-AcGFP. We mixed MDCK Lifeact-DsRed cells with MDCK AG1-Rac1N17 cells at a ratio of 1:99 and performed fluorescent live cell imaging. Images were edited using IMARIS software (Carl Zeiss, Oberkochen, Germany).
7
3D Reconstruction of Confocal Images
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Serial images (n = 50–100) were captured using a Mete confocal laser scanning microscope (CLSM; Carl Zeiss). The images were assembled and 3D reconstruction was performed using Imaris software (Carl Zeiss).
8
X-Ray Micro-CT Imaging of Zebrafish
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WT and stm mutant adult zebrafish were fixed with 70% ethanol and stored in 70% ethanol. The heads of zebrafish were scanned using an X-ray micro-CT device (ScanX- mate-E090S105, Comscantechno Co., Ltd., Japan) at a tube voltage peak of 60 kVp and a tube current of 100 μA. The sample was rotated 360° in steps of 0.24°, generating 1500 projection images of 992 × 992 pixels. The micro-CT data were reconstructed at an isotropic resolution of 13.3 × 13.3 × 13.3 μm. Three-dimensional tomographic images were obtained using OsiriX MD software (version 9.0, Pixmeo, SARL, Switzerland) and Imaris software (version 9.1, Carl Zeiss Microscopy Co., Ltd.). Supplementary video (Supplementary video 1, see section on supplementary materials given at the end of this article) was edited using Adobe Premiere Pro CC (Adobe Systems Co., Ltd., Japan).
9
Micro-CT Analysis of Male Pupal Head
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A male head tissue at 24 hours after pupal-chamber formation (24 h APF) was fixed in Carnoy solution at room temperature overnight, washed in 70% ethanol and stored in 70% ethanol. The sample was rehydrated through a graded ethanol series, and stained with 25% Lugol solution [64 (link)–66 (link)] for 5 days. The stained sample was scanned using an X-ray micro-CT device (ScanXmate-E090S105, Comscantechno Co., Ltd., Japan) at a tube voltage peak of 60 kVp and a tube current of 100 μA. The sample was rotated 360 degrees in steps of 0.24 degrees, generating 1500 projection images of 992 × 992 pixels. The micro-CT data were reconstructed at an isotropic resolution of 13.3 × 13.3 × 13.3 μm, and converted into an 8-bit tiff image dataset using coneCTexpress software (Comscantechno Co., Ltd., Japan). Three-dimensional tomographic images were obtained using the OsiriX MD software (version 9.0, Pixmeo, SARL, Switzerland) and Imaris software (version 9.1, Carl Zeiss Microscopy Co., Ltd., Japan). Supplemental videos were edited using Adobe Premiere Pro CC (Adobe Systems Co., Ltd., Japan).
10
Visualizing S. mutans Biofilm Formation
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2.1 | Biofilm-formation assay using fluorescence microscopy S. mutans MT8148 bacteria were grown at 37°C to an OD of 1.0 at 620 nm. Ten microliters of S. mutans was incubated with 0, 2, 10, or 50 µM sulfated vizantin in 2 mL of BHI medium supplemented with 1% sucrose (BHI-S) at 37°C for 72 hr in glass-bottom dishes (MatTek Corporation, MA). The culture medium was then replaced twice daily. The glass-bottom dishes were washed three times with phosphate-buffered saline (PBS; Nacalai Tesque, Kyoto, Japan). Then, live cells in each S. mutans biofilm were stained with 1 mL of calcein-AM (Molecular Probes, OR). After a 2 hr incubation at 37°C, the glass-bottom dishes were washed three times with sterile Dulbecco's modified Eagle medium (DMEM; Thermo Scientific, MA, USA). Next, the S. mutans biofilms were stained with 1 mL rhodamine B (Wako Pure Chemical Industries, Osaka, Japan). After a 5 min incubation at 37°C, the glass-bottom dishes were washed three times with sterile DMEM. Finally, the samples were fixed with mounting medium (Vector Laboratories, Inc., CA) and examined under a confocal laser-scanning microscope (LSM700, Zeiss, Germany). Images were drawn using Imaris software (Zeiss, Germany), and the thicknesses and densities of the S. mutans biofilms on the glass-bottom dishes were calculated using MetaMorph software (Molecular Devices, CA).
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