HeLa cells grown in glass chambers (Lab-Tek II) (Nalge Nunc Int.) were transfected with plasmids encoding Flag-wt TRIF, HA-(Δ160-181) TRIF, or HA-Stat4 proteins using TurboFect (Thermo Scientific, Pittsburg, PA) according to the manufacturer's instructions . Methanol-fixed cells were stained with fluorophore-conjugated anti-Flag (Alexa Fluor AF555) (3768) (Cell Signaling Technologies, Beverly, MA), and anti-HA (Alexa Fluor AF488) (2350) (Cell Signaling Technologies, Beverly, MA) antibodies. Nucleus staining was performed with DAPI. Confocal images of cells were acquired using a Zeiss LSM510 META laser-scanning microscope (Carl Zeiss MicroImaging, Thornwood, NY) equipped with a 40X C-apochromat objective lens (N.A.1.2). Image z-stacks were collected with consistent settings including an optical slice thickness of 1.0 μm, 0.11 μm x-y pixel size, and 0.40 μm z-axis step size. Maximum intensity projections (MIP) of each image stack were calculated using the LSM Image Browser software (v 4.0). Alternatively, HeLa cells were co-transfected with expression constructs encoding mCherry-wt TRIF and eGFP-(Δ160-181) TRIF fusion proteins. Cells were kept in a humidified, C02-fed stage-top chamber, and live cell imaging was acquired using the Zeiss LSM510 META laser-scanning microscope with images collected using the same confocal optical slice parameters.
Lsm 510 meta laser scanning microscope
Manufactured by Zeiss
Sourced in Germany, Italy
The LSM 510 META is a laser scanning microscope developed by Zeiss. It utilizes multiple laser sources to generate images by scanning a focused laser beam across a sample and detecting the emitted light. The microscope is designed for high-resolution imaging and analysis of biological and material samples.
Automatically generated - may contain errors
Lab products found in correlation
Turbofect, by Thermo Fisher Scientific (4 mentions)
Lsm 510, by Zeiss (3 mentions)
Poly l lysine (pll), by Merck Group (2 mentions)
Vectashield, by Vector Laboratories (2 mentions)
Zen software, by Zeiss (2 mentions)
Bx61 microscope, by Olympus (2 mentions)
Lsm 5 image browser, by Zeiss (2 mentions)
Zo1 1a12, by Thermo Fisher Scientific (2 mentions)
Anti vinculin, by Abcam (2 mentions)
Plan apochromate 63 1.4 oil immersion objective, by Zeiss (2 mentions)
Prolong gold mounting medium, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 647 conjugated wheat germ agglutinin, by Thermo Fisher Scientific (2 mentions)
Lsm 780, by Zeiss (2 mentions)
Zen blue software, by Zeiss (2 mentions)
Lsm software, by Zeiss (1 mentions)
Proximity probes, by Olink (1 mentions)
In cell analyzer, by GE Healthcare (1 mentions)
Plasmid mega kit, by Qiagen (1 mentions)
Normal serum, by Santa Cruz Biotechnology (1 mentions)
Triton x 100, by Merck Group (1 mentions)
68 protocols using lsm 510 meta laser scanning microscope
1
Fluorescence Microscopy of TRIF Signaling Dynamics
2
Immunofluorescence Staining of Permeabilized Cells
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Cells were seeded on glass cover-slips coated with poly-L-lysine (Sigma-Aldrich, catalog number P4707) for confocal imaging. Cells were perforated with 0.5% β-escin, which is a naturally derived saponin mixture, diluted with a solution of 80 mM PIPES, pH 6.8, supplemented with 5 mM EGTA and 1 mM MgCl2 for 5 min and fixed with 3% paraformaldehyde–PBS for 15 min as described previously [51 (link)]. Non-specific binding was blocked with 5% normal serum from the host animal used to generate the secondary antibody (Jackson ImmunoResearch Laboratories; West Grove, PA). Cells were stained with primary or secondary antibodies overnight at 4°C or 1 h at room temperature. Antibodies were diluted in PBS supplemented with 0.5% β-escin and 3% radioimmunoassay-grade BSA. Single vertical confocal images were acquired with a Zeiss LSM 510 Meta laser scanning microscope (Carl Zeiss MicroImaging, Jenna, Germany) using diode (excitation 405 nm), Argon (excitation 488 nm), and HeNe (excitation 543 and 633 nm) lasers, 40× or 100× Plan Apo NA 1.4 objectives, and Zeiss LSM software (Carl Zeiss MicroImaging, Jenna, Germany). Confocal images were overlayed with phase contrast images to draw cell and nucleus outlines using graphics software. Quantification of co-localization was performed by measurement of Mander's coefficient in at least 10 cells per experiment using ImageJ.
3
Quantitative Proximity Ligation Assay
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The cells were washed and allowed to react with the following pairs of proximity probes (Olink Bioscience): rabbit anti-ASC and mouse anti-phosphotyrosine, rabbit anti-p-FAK and mouse anti-mCherry, and anti-p-Pyk2 and mouse anti-mCherry. In situ PLA was performed using the Duolink Proximity Ligation in situ reagent kit (Olink, Uppsala, Sweden), according to the manufacturer’s instructions. The results were visualized under confocal microscopy using a Zeiss LSM510 META laser scanning microscope (Carl Zeiss, Germany) with a 63 × 1.32 or 100 × 1.32 NA oil immersion objective. Quantification was performed with an IN Cell Analyzer (GE Healthcare).
4
Arabidopsis and Maize Protoplast Transformation
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Arabidopsis mesophyll protoplasts were isolated [74] (link) and transformed using polyethylene glycol (PEG4000). Approximately 1.5×105 protoplasts were transformed with 10 mg of plasmid DNA per test construct for single protein expression. Maize leaf protoplasts were isolated from etiolated maize seedlings as described in Sheen and Bogorad [75] (link) and transformed using PEG4000 [76] (link). Approximately 3×105 protoplasts were transformed with 12 mg of plasmid DNA per test construct for single protein expression. Plasmid DNA was prepped using the Qiagen (Germantown, MD) Plasmid Mega kit. Protoplasts were incubated a total of 18 to 24 hours at 22°C and analyzed for GFP fluorescence using confocal microscopy. Confocal fluorescence microscopy analysis was performed using a Zeiss LSM510 META Laser Scanning Microscope (Carl Zeiss MicroImaging, Inc., Thornwood, NY) equipped with a Krypton-Argon Ion (488 nm) laser, a green (543 nm) Helium-Neon laser, FITC, and Texas red filter sets. Image acquisition and analysis was performed using LSM 5 Image Examiner, version 4.2 (Carl Zeiss MicroImaging, Inc.), using a 40x water 1.2 numerical aperture objective. Excitation wavelengths used were 488 nm and 543 nm, and emission filters were 500 to 530 nm and 630 to 700 nm to detect GFP and chlorophyll, respectively.
5
Visualizing α1A-AR trafficking dynamics
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HEK293 cells were transiently co-transfected with FLAG-tagged α1A – AR and one of the following GFP-tagged proteins: Rab5 (Rab5 Q79L) variant, Rab11 (Rab11 S25N) variant, β-arrestin-1 or β-arrestin-2. Rab5 and Rab11 were kindly provided by Dr. Marino Zerial (Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany). Following serum-deprivation for 24h, cells were stimulated with 1 μM A-61603 or 1 mM ISO for 2h. After treatment, cells were fixed with freshly prepared 3.7% paraformaldehyde in PBS for 15 min at room temperature. Subsequently, cells were permeabilized with 0.1% Triton X-100 (Sigma-Aldrich) in PBS for 5 min, followed by nonspecific binding site blocking with 3% normal serum (Santa Cruz Biotechnology) in PBS for 1 h. Incubation with Alexa Fluor-568 conjugated anti-FLAG antibodies in blocking solution was done as directed by the manufacturer. Confocal microscopy was performed using a Zeiss LSM 510 META laser scanning microscope (Carl Zeiss, Inc., Thornwood, NY) equipped with a 60X objective, using the following laser wavelengths: excitation at 488 nm and emission at 505–530 nm; excitation at 543 nm and emission at 560–615 nm.
6
Immunostaining of Cortical Neurons
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Immunostaining of rat cortical neurons (E18, DIV14) was performed as previously described (Woo et al. 2007 (link)). Briefly, neurons were fixed with 4% paraformaldehyde and 4% sucrose in PBS for 20 min. The cells were permeabilized by incubation in PBS containing 1% BSA and 0.1% Triton X-100 for 30 min at room temperature. After washing, cells were incubated in buffer containing antibodies against EAAC1 (1:100) at 4 °C overnight and were then incubated with FITC-conjugated AffiniPure goat anti-mouse IgG (Jackson ImmunoResearch Laboratories, Inc., 1:200) in buffer for 2 h at room temperature. Nuclei were counterstained with Hoechst (10 μM in PBS) for 30 min. Stained cells were mounted in VECTASHIELD (Vector Laboratories) and observed under a Zeiss LSM 510 META laser scanning microscope (Carl Zeiss, Germany).
7
Microscopic Imaging of Cell Populations
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Immunhistochemical staining was documented using a Zeiss Axiophot microscope (Carl Zeiss MicroImaging, Jena, Germany) or a Zeiss LSM510 Meta laser scanning microscope (Zeiss, Oberkochen, Germany). For determining cell numbers, the 10x objective was used. Images were adjusted for contrast in AxioVision LE Rel. 4.3 (Carl Zeiss MicroImaging).
8
Confocal Imaging of Ovule Development
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The confocal observation of ovules was performed according to the method described previously (Christensen et al., 1997 (link)) with slight modifications. Inflorescences were harvested and fixed in 4% glutaraldehyde (in 12.5 mM cacodylate, pH 6.9), and a vacuum was applied for the initial 20 min, after which they were in fixative overnight at room temperature. After fixation, the tissues were dehydrated through a conventional ethanol series with 20 min per step. After the dehydration, the tissue was cleared in 2:1 (v/v) benzyl benzoate: benzyl alcohol for a minimum of 1 h. Pistils or siliques were dissected, mounted with immersion oil, and observed with a Zeiss LSM510 META laser scanning microscope (Zeiss, Jena, Germany) with a 488-nm argon laser and an LP 530 filter. Images were edited with Zeiss LSM Image Browser software and Photoshop software. The central cell vacuole size was calculated according to the method as described previously (Wu et al., 2012 (link)). Single section image was captured when scanning to the largest sectional view of the central cell vacuole, and the size of central cell vacuoles were calculated using Image J software.
9
Quantifying Sister Chromatid Exchange in ESCs
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BLM+/+ and BLM−/− ESCs were grown to 80% confluence before they were treated with 10 μM BrdU (Thermofisher) for 48 hours, after which mitotic arrest was induced with 150 ng/ml Colcemid (Gibco) for 30 minutes. Cells were then incubated in 75 mM KCl for 15 minutes at 37°C before fixation in a solution of 3 parts methanol (Sigma-Aldrich) to 1 part glacial acetic acid (Sigma-Aldrich). The cell suspension was dropped onto pre-chilled slides, counterstained with 0.1 mg/ml acridine orange (Molecular Probes), and mounted in Sorenson Buffer, pH 6.8 (0.1 M Na2HPO4 (Sigma-Aldrich), 0.1 M NaH2PO4 (Sigma-Aldrich)). Chromatids were visualized using the Zeiss LSM 510 Meta Laser Scanning Microscope (Carl Zeiss) under the FITC filter. SCE events in a metaphase spread was counted and normalized to the total number of chromosomes.
10
Collagen Fiber Orientation Mapping in Vascular Tissues
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To image collagen fibers in both controls and explants, the CNA35-OG488 fluorescent probe was used. 7 Samples were incubated overnight with a 3 mM CNA probe. Subsequently, samples were washed thrice with PBS and placed in chamber slides for imaging with a Zeiss LSM 510 Meta laser scanning microscope (Carl Zeiss) coupled to an inverted Axiovert 200 microscope (Carl Zeiss). To evaluate the orientation in different regions of the vascular wall, tile scans in the x-y plane were made at different locations along the z direction (=along the radial direction of the vascular wall and up to maximum 200 mm from both the luminal and outer side) by programming the computerized x-y stage. The x-direction and y-direction were defined parallel to the circumferential and the axial direction of the wall, respectively, which were marked upon excision of the samples. Tiles were stitched together in MATLAB (Mathwork) to reconstruct x-y surfaces. 18
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