To determine the percentage of cells with aggregated (A), partially aggregated (PA) or dispersed (D) Met, GFP alone or EB3‐GFP transfected cells were stimulated with 0.5 nM HGF for 20 minutes and fixed with formaldehyde. The Met‐positive vesicles “at the MTs plus‐ends” have been scored at the vicinity of +end, stained with EB3‐GFP. The number of cells with aggregated, partially aggregated or dispersed Met were determined as previously described.33 Briefly, cells in each category have been counted under a scanning confocal microscope (LSM 510 Meta laser; Carl Zeiss, Inc.) with a ×100 plan Apo 1.4 NA objective and driven by ZEN LE software (Carl Zeiss, Inc.). In each experiment, a minimum of 20 fields was scored. See Figure S3D for representative images of each cell types.
Lsm 510 meta laser
Manufactured by Zeiss
Sourced in Germany
The LSM 510 Meta is a laser scanning microscope designed and manufactured by Zeiss. It is capable of performing multi-photon excitation and spectral imaging. The system utilizes a tunable femtosecond pulsed laser as the excitation source.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 488, by Thermo Fisher Scientific (4 mentions)
Zen le software, by Zeiss (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (1 mentions)
Fluorescent mounting medium, by Agilent Technologies (1 mentions)
Mms 101p, by Fortrea (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Mowiol, by Merck Group (1 mentions)
15 mm glass coverslips, by Thermo Fisher Scientific (1 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (1 mentions)
Axiovert 200 microscope, by Zeiss (1 mentions)
Zen software, by Zeiss (1 mentions)
Dmirb inverted microscope, by Leica (1 mentions)
Dm irb inverted microscope system, by Leica (1 mentions)
Dapi mounting media, by Vector Laboratories (1 mentions)
Te300, by Nikon (1 mentions)
Alexa fluor 568, by Thermo Fisher Scientific (1 mentions)
Lsm imaging software, by Zeiss (1 mentions)
Anti ha high affinity, by Roche (1 mentions)
24 protocols using lsm 510 meta laser
1
Quantifying Subcellular Met Localization
2
Germ Cell Morphology Quantification
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Gonads were dissected in M9 with 0.25 mM levamisole and 100 ng/mL Hoechst 33342 (Invitrogen). Images were collected using a 63× objective on a Zeiss LSM510 Meta laser scanning confocal microscope. Full projections of z-stacks containing approximately twenty 1 µm sections were made using ImageJ [1.41o] [52] . Core projections were made using the central ten 1 µm sections that did not include the top and bottom surfaces of the cap. The lengths of the cap and LEPs were measured using the full maximum intensity projection and counting the number of germ cell nuclei along the distal-proximal axis to the proximal edge of the cap and longest LEP. The plexus was measured using the core projection and counting the number of germ cell nuclei to the point where fluorescence drops to background levels.
3
Immunofluorescence Imaging of Transfected HeLa Cells
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1 x 105 HeLa cells grown on coverslips (Marienfeld, Lauda Königshofen, Germany) were transfected with 500 ng A4 expression plasmids by applying Lipofectamine LTX transfection reagent. At day two post transfection, cells were fixed in 4% paraformaldehyde in PBS for 30 min, permeabilized in 0.1% Triton X-100 in PBS for 45 min, incubated in blocking solution (10% donkey antiserum (Sigma-Aldrich) in PBS) for 1 h, and treated with anti-HA Ab (MMS-101P; Covance) in blocking solution for 1 h. Donkey anti-mouse Alexa Fluor 488 (Life Technologies) was used as secondary Ab in a 1:300 dilution in blocking solution for 1 h. Finally, nuclei were stained using DAPI (4’, 6’-diamidino-2-phenylindole; 1:1000 in PBS) (Merck Millipore, Darmstadt, Germany) for 5 min. coverslips were mounted on glass microscope slide (Marienfeld) using Fluorescent Mounting Medium (DAKO, Hamburg, Germany). The images were captured by using a 63x objective on a Zeiss LSM 510 Meta laser scanning confocal microscope. x-z optical sections were acquired from 0.28 μm layers.
4
Multicolor Fluorescence Microscopy Protocol
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Imaging was performed on a Zeiss LSM-510 META laser scanning microscope using a Zeiss 63X 1.4 numerical aperture oil immersion lens. The optical section thickness was typically 1 micron. mTagBlue was imaged using a 405 nm laser and a BP 390-465 filter. ECFP fluorescence was imaged using 458 nm laser excitation source and a BP 475-525 filter set. EGFP and YFP fluorescence was visualized using a 488 nm excitation laser and a 500-530-nm emission filter set. mRFP fluorescence was imaged using a 543 nm excitation laser and BP 565-615 filter set. Alexa Fluor 647 fluorescence was imaged using 647 nm excitation laser, and a LP 650 filter. The approximate outline of each cell was traced by hand using Adobe Illustrator using the surface labeled HA-fluorescence as a guide; when this was indistinct, the outlines were derived from images which were saturated to show cell edges.
5
Immunofluorescence Staining of Stimulated Cells
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Cells seeded onto coverslips were stimulated with anti-CD3 for the time indicated and fixed with 4% paraformaldehyde for 30 min at room temperature, followed by permeabilization with 0.1% Triton X-100 for 5 min, and blocked with 10% FBS for 30 min at room temperature. Cells were then incubated with primary antibodies in 5% FBS at 4 °C overnight. After washing three times (10 min each) with PBS, cells were incubated for 30 min with secondary antibody conjugated with fluorescence at 37 °C. The specimens were analyzed using a Zeiss (Oberkochen, Germany) LSM 510 Meta laser scanning confocal microscope system.
6
Viral Infection Localization in Mice
7
Calcium Imaging of GABAergic Neurons
8
Immunofluorescence Staining of Microtubules
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Cells grown on collagen‐coated coverslips were fixed in 4% formaldehyde at room temperature or 100% methanol at −20°C for MTs staining and permeabilized in 0.2% Triton X‐100 before the addition of antibodies. Antibodies used for IF were against the following: Met (AF276 from R&D Systems); α‐tubulin (Boehringer, Ingelheim), CLIP‐170, GFP mouse (Roche, Inc.). Secondary antibodies and Alexa Fluor 488, 546 and 633, and phalloidin were obtained from Molecular Probes. Images were recorded with a scanning confocal microscope (LSM 510 Meta laser; Carl Zeiss, Inc.) with a ×100 plan Apo 1.4 NA objective and driven by ZEN LE software (Carl Zeiss, Inc.).
9
Mitochondrial Staining of C6-Treated Cells
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The cells were seeded at a 1 × 103 cells per well in a µ-slide (Ibidi, Martinsried, Germany). After overnight attachment, the cells were treated with C6 (40 µM). The spent media were removed, and cells were stained using the CytoPainter Orange mitochondrial staining kit (1:500), as per the manufacturer’s instructions. After a 30 min incubation, the stain was replaced with binding buffer and the cells were imaged with the Zeiss LSM510 Meta laser scanning confocal microscope using the DAPI and TRITC filter sets.
10
Immunofluorescence Staining of CCTα and LMNA/C
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Cells cultured on 1-mm glass coverslips were fixed with 4% (wt/vol) paraformaldehyde in PBS for 15 min and permeabilized with 0.05% Triton X-100 in PBS at 4°C for 15 min. Coverslips were blocked overnight at 4°C in PBS containing 1% (wt/vol) BSA (PBS/BSA). CCTα (1:1000 dilution), CCTα-pS315+pS319 (1–2 µg/ml), CCTα-pY359+pS362 (1 µg/ml), and LMNA/C (1:250–1:500 dilution) antibodies in PBS/BSA were incubated with coverslips on parafilm in a humidity chamber for 4 h at room temperature or overnight at 4°C. After incubation with Alexa Fluor–conjugated secondary antibodies, coverslips were mounted on glass slides using Mowiol (Sigma-Aldrich) and cells were imaged using a Zeiss LSM 510 Meta laser scanning confocal microscope equipped with a 100× Plan APOCHROMAT 100×/1.40 numerical aperture oil immersion objective and argon 488/HeNe 548 lasers. The confocal images shown in figures were representative of results from three or four independent experiments.
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