At the eight-cell stage, HaloTag N-cadherin mRNA (500 pg/embryo) and membrane-mCherry mRNA (300 pg/embryo) were injected, and the embryos were cultured until stage 19. HaloTag-N-cadherin–expressing NC explants were dissected and preincubated for 30 min at room temperature with Alexa Fluor 488–nonpermeable HaloTag ligand solution in 1× Marc’s modified Ringer (MMR; 1:1,000, Promega; Los and Wood, 2007 (link)), and the excess ligands were washed. Then labeled NC cells were cultured on fibronectin-coated coverslips (Tissue Tek II; Nalgene), and time-lapse imagining was performed (LSM 510 Meta; Carl Zeiss) immediately after cell attachment. The lens used for this imaging was a Plan-Apochromat 63×/1.4 oil objective. The temperature was ∼23°C. The imaging medium was Immersol 518F (Carl Zeiss). Images were obtained and analyzed with LSM 510 version 3.2 microscope operation software and Zeiss Image Browser (both from Carl Zeiss).
Immersol 518f
Manufactured by Zeiss
Sourced in Germany, United States
Immersol 518F is a high-refractive-index immersion oil produced by Zeiss. It is designed for use in microscopy applications that require a specific refractive index to match the optical properties of the sample being observed. The product specifications and performance characteristics are provided by the manufacturer.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 710, by Zeiss (6 mentions)
Axiovert 200 inverted microscope, by Zeiss (4 mentions)
Efficient navigation 2, by Zeiss (4 mentions)
Lsm 780, by Zeiss (4 mentions)
Lsm 880, by Zeiss (4 mentions)
Lsm 880 confocal microscope, by Zeiss (4 mentions)
Plan neo 100 1, by Zeiss (3 mentions)
Plan apo 63 1, by Zeiss (3 mentions)
Prolong gold antifade, by Thermo Fisher Scientific (3 mentions)
Hoechst dye, by Merck Group (3 mentions)
Lsm 780 confocal microscope, by Zeiss (3 mentions)
Axio observer z1, by Zeiss (3 mentions)
Lumar v12 stereomicroscope, by Zeiss (3 mentions)
Plan neofluor 20, by Zeiss (2 mentions)
Orca flash 4.0 lt scmos camera, by Hamamatsu Photonics (2 mentions)
Apotome2 grid confocal unit, by Zeiss (2 mentions)
Plan apochromat 40 0.95 na air, by Zeiss (2 mentions)
Plan apochromat 63 1.40 na oil, by Zeiss (2 mentions)
Axio imager m2 microscope, by Zeiss (2 mentions)
Dmi6000b epifluorescence inverted microscope, by Leica camera (2 mentions)
42 protocols using immersol 518f
1
Visualizing N-cadherin Dynamics in Neural Crest Cells
2
Immunofluorescence Staining Protocol
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For p50, p150glued and pAKT immunofluorescence, cells were fixed in 3% paraformaldehyde followed by permeabilization with 0.2% Triton‐X‐100 for 10 min. Nuclei were visualized using Hoechst dye (33258; Sigma‐Aldrich). Coverslips were mounted on glass slides using ProLong Gold Antifade (Invitrogen). Cells were visualized with an Axiovert 200 inverted microscope (Carl Zeiss, Inc.) using Plan‐Neo 100×/1.30 or Plan‐Apo 63×/1.40 oil‐immersion objectives (Immersol 518F; Carl Zeiss, Inc.). In some cases, optical sections were deconvolved using axiovision combined iterative algorithm to obtain confocal images.
3
Fluorescent Imaging of Lysate Granules
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Fluorescent imaging was performed at room temperature using a Yokogawa CSU W1 spinning disk attached to a Nikon Ti2 eclipse with a Photometrics Prime 95B camera using Nikon Elements software (versions 5.20.00 to 5.21.02). Imaging was performed through a Nikon Plan Apo 60× 1.40 NA oil objective with Immersol 518 F (Zeiss; refractive index 1.518), and Perfect Focus 2.0 (Nikon) was engaged for all captures. For single time point captures using the Ibidi Angiogenesis slides, images were taken at the surface of the sample at 30 min after induction or at the indicated time point. Imaging was performed using 488-nm, 555-nm, and 640-nm lasers when applicable along with a capture of DIC. Multipoint time-lapse imaging was performed in Lab-Tek chambered cover glass slides, where 5 xy fields were stored for each condition. Images were taken at each xy position every 10 min. Automated granule detection and measurement were performed using CellProfiler software similar to the methods described previously (Mackenzie et al., 2017 (link)), excluding association of granules to cells. Briefly, the lysate granules were segmented by applying a global minimum cross entropy approach on the GFP channel. The sizes represent an average of at least five separate fields, with each field containing at least 100 segmented granules.
4
Perfusion-Based Tissue Preservation and Imaging
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Images shown in all figures are representative of data acquired from at least N = 3 animals per experiment. Animals under deep isoflurane anesthesia were perfused transcardially with ice-cold PBS, followed by 4% paraformaldehyde (PFA) in 0.1 M PBS (pH 7.4). Before sectioning, tissues were cryoprotected by immersion overnight in 15% sucrose, followed by 24 h in 30% sucrose in PBS. Tissues were then frozen in OCT compound (Fisher) using a beaker of 2-methylbutane chilled in dry ice. Ten- or 50-µm-thick cryosections were stored at −80°C until use. Whole brains and hearts were imaged immediately after dissection using an Olympus SZX-12 with an SZX-RFL2 coaxial fluorescence attachment. Cryosections were imaged using a Leica TCS SP8X confocal microscope equipped with LAS X software and a 63× oil immersion objective (1.4 N/A). Some images were obtained using a Zeiss Axiovert 200 inverted microscope equipped with AxioVision software and a Plan-Neo 100 Å∼/1.30 and Plan-Apo 63 Å∼/1.40 oil-immersion objectives (Immersol 518F; Carl Zeiss, Inc.) or a Plan-Neofluor 20× dry objective.
5
Fluorescent Imaging of Fat Cells and Nephrocytes
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Fluorescent images were obtained at RT with an AxioImager.M2 microscope (Carl Zeiss, Oberkochen, Germany) with an ApoTome2 grid confocal unit (Carl Zeiss) using EC Plan-Neofluar 40×/0.75-NA Air (Carl Zeiss) or Plan-Apochromat 40×/0.95-NA Air (Carl Zeiss) objectives for fat cells, and Plan-Apochromat 63×/1.40-NA Oil (Carl Zeiss) objective for nephrocytes, an Orca Flash 4.0 LT sCMOS camera (Hamamatsu Photonics, Hamamatsu, Japan), and Zeiss Efficient Navigation 2 software (Carl Zeiss). Immersol 518F (Carl Zeiss) immersion oil was used for the 63x objective. Images from 8 consecutive focal planes (section thickness: 0.25 µm for nephrocytes and 0.35 µm for fat cells) were projected onto one single image, except for the colocalization assays, where we aimed to exclude any false positive colocalization, thus assessing only one focal plane. Images were processed in Zeiss Efficient Navigation 2 (Carl Zeiss) and Photoshop CS4 or CS6 (Adobe, San Jose, CA, USA) to present final figures.
6
Mitochondrial Imaging with MitoTrackers
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Measurements were performed in thin-bottom 8-well chambered cover glass (Thermo Fisher Scientific, USA, 155409), using an apochromate objective (420792-9800-720, Zeiss) with oil immersion (ISO 8036, Immersol 518F, Zeiss).
MitoTrackers were excited by 561 nm laser for MitoTrackers Orange-CMTMRos and Red-CMXRos, and by 633 nm laser for Deep Red FM. Emission was recorded in λ-mode with 34-channel QUASAR detector unit from Zeiss. All images were processed using Zen 2012 (Zeiss, Germany) and Fiji53 (link) software. Supplementary Video2 was drift corrected using a StackReg -plugin for Fiji54 (link).
MitoTrackers were excited by 561 nm laser for MitoTrackers Orange-CMTMRos and Red-CMXRos, and by 633 nm laser for Deep Red FM. Emission was recorded in λ-mode with 34-channel QUASAR detector unit from Zeiss. All images were processed using Zen 2012 (Zeiss, Germany) and Fiji53 (link) software. Supplementary Video
7
Cytological Analysis of Arabidopsis Flowers
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Images were taken using Leica DMI6000b epifluorescence inverted microscope, equipped with YFP, DAPI and DsRed filter cube. For YFP expression analysis, dissected sepals were transferred to 10% glycerol and data were collected. For chromosome spread, data from DAPI stained samples were collected with the aid of 100 × oil objective and an immersion oil Immersol 518F (Zeiss, Germany) or by using confocal laser scanning microscopy with Airyscan module (Zeiss LSM 880) at excitation of 405 nm and emission of 421 nm. CENH3-GFP localization was observed at excitation of 488 nm and emission of 510 nm. 7 DAP silique images were captured by using Leica S8apo stereomicroscope.
8
Fabrication of Magnetically Actuated Microstructures
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The fabrication platform of the MAC consists of a commercially available 2PP-based direct laser writing system (Photonic Professional, Nanoscribe GmbH, Germany) and a 5-coil electromagnetic coil setup (fig. S1 ) (45 ). A 63× 1.4 numerical aperture oil-immersion objective (Carl Zeiss AG, Germany) was used for 3D microprinting the MAC. A refractive index matching oil (Immersol 518 f; Carl Zeiss Microscopy GmbH) was applied onto the objective to successfully detect the SF solution–substrate interface. The laser power of the 2PP system was 50 mW, which produced a printing power of 20, 30, 40, and 50 mW at a laser intensity of 40, 60, 80, and 100%, respectively. Unless otherwise specified, the laser scanning speed was kept constant at 10,000 μm s–1.
9
Fluorescent In Situ Hybridization of Sulfurimonas
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FISH was performed on filters with fluorescently-labeled oligonucleotides (Glöckner et al., 1996 (link)). EPSY549 (5′-CAGTGATTCCGAGTAACG-3′) was labeled with Atto RHO 101 and used to target Epsilonproteobacteria (Lin et al., 2006 (link)). EPSY549Mod (5′-CAGTGATTCCGAATAACG-3′) was modified to target the identified Sulfurimonas phylotype and labeled with Atto RHO 101. NONEUB338 probes were labeled with Atto RHO 101 and used as control for non-specific staining (Christensen et al., 1999 (link)). Hybridizations were performed at 30% formamide for EPSY549 and at 20% formamide for EPSY549Mod. Fixed cells of Escherichia coli were used as a negative control. After the in situ hybridization, washing and drying, the cells were stained with the fluorescent DNA-binding dye 4′,6-diamidino-2-phenylindole (DAPI) (Morikawa and Yanagida, 1981 (link)). Stained slides were immersed in Immersol 518F (Carl Zeiss AG, Oberkochen, Germany) and evaluated in Zeiss Axio Imager Z1 microscope (Carl Zeiss Microscopy GmbH, Göttingen, Germany), equipped with filter 49 (DAPI), and 64 HE mPlum (Atto RHO 101).
10
Quantification of Nuclear NF-κB p65
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Eight-well μ-slides (Ibidi) were coated with poly-l -lysine (Sigma) before BMDMs were seeded at 2 × 105 cells/well and experiments were performed. Cells were then fixed with 4% paraformaldehyde for 30 min, before blocking and permeabilizing cells (IF buffer: PBS, 10% FBS, 0.5% Triton X-100) for 1 h, before staining with a rabbit monoclonal anti-NF-κB p65 (Cell Signaling Technology; clone C22B4, 4764, 1:100) overnight at 4 °C. Cells were then stained with a secondary donkey anti-rabbit Alexa-488 antibody (Thermo Scientific; R37118, 1:1000) for 1 h at room temperature, before nuclear staining with 4′,6-diamidino-2-phenylindole (1 μm ) for 5–10 min. After each step, cells were washed 2–3 times with IF buffer or PBS. Cells were imaged using a Zeiss LSM 780 confocal microscope; 2 × 2 tile scans were obtained for each experimental condition using a ×40 oil objective with Immersol 518 F (Zeiss) and acquired with ZEN 2012 version 8.1 software (Zeiss). Images were generated as tagged image bitmap files (TIFF) using FIJI software. Quantification of nuclear NF-κB p65 was performed using FIJI software by overlaying nuclei identified with 4′,6-diamidino-2-phenylindole staining (not shown) onto the Alexa-488 channel before measurement of mean fluorescent intensity within each nucleus.
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