For morphological characterization, eye primordia were imaged after 24 hours in culture by phase contrast microscopy on a Nikon Eclipse TE2000-U or a Zeiss AxioObserver A1 microscope (10x Ph1, NA = 0.3). For time-lapse imaging of axon growth, eye primordia were imaged after 18–24 h in culture using a Nikon Eclipse TE3000 microscope (10x Ph1 objective, NA = 0.25). Images were captured every 30 seconds for ∼3 h using a CCD camera (AxioCam ERc 5s ; Zeiss, UK) and ZEN 2011 software. For turning assays (see below), axons were imaged after 12-24 hours in culture using a Nikon Eclipse TE3000 (20x Ph1, NA = 0.45). Images were captured every 30 seconds using a Hamamatsu c4742-95 camera (Hamamatsu Photonics, Japan) and OpenLab software.
Eclipse te3000
Manufactured by Nikon
Sourced in Japan
The Eclipse TE3000 is a research-grade inverted microscope designed for advanced microscopy applications. It features a high-quality optic system, a motorized stage, and integrated image capture capabilities. The Eclipse TE3000 is a versatile platform suitable for a range of laboratory and research tasks.
Automatically generated - may contain errors
Lab products found in correlation
C4742 95, by Hamamatsu Photonics (2 mentions)
Axiocam erc5, by Zeiss (2 mentions)
Eclipse te2000 u, by Zeiss (2 mentions)
Axio observer a1 microscope, by Zeiss (2 mentions)
Ems3 sycop3, by Zeiss (2 mentions)
Plan neofluor z objective, by Zeiss (2 mentions)
Prime bsi express camera, by Teledyne (2 mentions)
Csu x, by Yokogawa (2 mentions)
Gridded scoring dish, by STEMCELL (2 mentions)
Lsm 510, by Zeiss (2 mentions)
Methocult sf m3236, by STEMCELL (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Nichirei Biosciences (1 mentions)
Dxm1200, by Nikon (1 mentions)
Ix2 dsu, by Olympus (1 mentions)
Orca flash 4, by Hamamatsu Photonics (1 mentions)
Fv1000 clsm, by Olympus (1 mentions)
Ox 42, by Bio-Rad (1 mentions)
Neuronal nuclei (neun), by Merck Group (1 mentions)
Prolonged gold, by Thermo Fisher Scientific (1 mentions)
9 protocols using eclipse te3000
1
Imaging Techniques for Axon Growth
2
Imaging Techniques for Axon Growth
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For morphological characterization, eye primordia were imaged after 24 hours in culture by phase contrast microscopy on a Nikon Eclipse TE2000-U or a Zeiss AxioObserver A1 microscope (10x Ph1, NA = 0.3). For time-lapse imaging of axon growth, eye primordia were imaged after 18–24 h in culture using a Nikon Eclipse TE3000 microscope (10x Ph1 objective, NA = 0.25). Images were captured every 30 seconds for ∼3 h using a CCD camera (AxioCam ERc 5s ; Zeiss, UK) and ZEN 2011 software. For turning assays (see below), axons were imaged after 12-24 hours in culture using a Nikon Eclipse TE3000 (20x Ph1, NA = 0.45). Images were captured every 30 seconds using a Hamamatsu c4742-95 camera (Hamamatsu Photonics, Japan) and OpenLab software.
3
Imaging Transgenic Larvae and Fungal Interactions
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Transgenic larvae were pre-screened for fluorescence using a zoomscope (EMS3/SyCoP3; Zeiss; Plan-NeoFluor Z objective). For multi-day imaging experiments, larvae were anesthetized and mounted in a Z-wedgi device [43 (link),48 (link)] where they were oriented such that the hindbrain was fully visible. Z-series images (5 μm slices) of the hindbrain were acquired on a spinning disk confocal microscope (CSU-X; Yokogawa) with a confocal scan head on a Zeiss Observer Z.1 inverted microscope, Plan-Apochromat NA 0.8/20x objective, and a Photometrics Evolve EMCCD camera. Between imaging sessions larvae were kept in E3-MB with PTU in individual wells of 24- or 48-well plates. Neutrophil-fungal interactions were imaged using an inverted epifluorescence microscope (Nikon Eclipse TE3000) with a Nikon Plan Fluor 20x/0.50 objective, motorized stage (Ludl Electronic Products) and Prime BSI Express camera (Teledyne Photometrics). Environmental controls were set to 37°C with 5% CO2. Images were acquired every 3 min for 12 h. Imaging of A. fumigatus stained with CFW was performed using an upright Zeiss Imager.Z2 LSM 800 laser scanning confocal microscope with Airyscan detection and a Plan-Apochromat 20x /0.8 objective. A single z plane image was acquired for each hypha. Images were captured using identical laser and exposure settings for each condition.
4
Enumeration of EPC Colonies from PBMNCs and BMMNCs
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Freshly isolated human or mouse peripheral blood mononuclear cells (PBMNCs), and mouse BM mononuclear cells (BMMNCs) were cultured in semisolid methyl cellulose-based culture medium, (MethoCult™ SF M3236, STEMCELL Technologies Inc., Vancouver, BC, Canada) containing 100 ng/mL SCF, 50 ng/mL VEGF, 50 ng/mL basic fibroblast growth factor (bFGF), 50 ng/mL epidermal growth factor (EGF), 50 ng/mL insulin-like growth factor (IGF), 50 ng/mL interleukin-3 (IL-3) (these six proteins were purchased from Peprotech, Inc. Rocky Hill, NJ, USA), 2 IU/mL heparin (Ajinomoto Pharmaceutical Co. Ltd. Tokyo, Japan), 30% (v/v) fetal bovine serum (Nichirei Biosciences Inc., Tokyo, Japan) and penicillin/streptomycin (100 U/100 μg/mL; Gibco). Cells were seeded at 1.5 × 105 cells/35 mm dish (BD Falcon, BD Bioscience, San Jose, CA, USA) and left in a humidified incubator with 5% CO2 at 37°C till EPC colony formation. The number of adherent colonies on the dishes was counted between day 6–10 (mouse) and 16–18 (human) using gridded scoring dish (STEMCELL Technologies Inc. Vancouver, BC, Canada) under a phase-contrast light microscope (Eclipse TE3000; Nikon, Tokyo, Japan). Primitive EPC colony-forming units (pEPC-CFUs) and definitive EPC colony-forming units (dEPC-CFUs) were separately counted [16 (link)].
5
Paraffin-Embedded Histological Sectioning
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For light microscopic investigations, prosomata of the above-listed species (except G. arabs) were fixed according to the method of Duboscq-Brasil [49 ]. After dehydration in ethanol, specimens were transferred to tetrahydrofuran and finally embedded in paraffin. Embedded specimens were serially sectioned (4–10 μm) with the rotary microtome Leica RM 2125 RT. Sections were stained with Azan according to the protocol of Geidies [49 ], and investigated and digitized with a Nikon Eclipse TE3000 microscope equipped with a Nikon DXM1200 camera.
6
Fluorescence Microscopy Imaging Protocol
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Fluorescent images were obtained using a fluorescent microscope (Eclipse TE3000, Nikon, Japan) with a CMOS camera (ORCA flash 4.0, Hamamatsu Photonics, Japan), and a laser scanning confocal microscope (LSM510, Carl Zeiss, Germany or IX81, Olympus, Japan). The microscope setup for the spinning disc superresolution microscope (SDSRM) is based on a disk-scanning confocal microscope system, which includes an IX81 and a disc-scanning unit (IX2-DSU, Olympus, Japan). To investigate neuritogenesis and F-actin bundle formation, acquired images were analyzed with ImageJ (NIH, http://rsb.info.nih.gov/ImageJ ).
7
Immunofluorescence Analysis of Spinal Cord
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A phosphorylated S6 (pS6)‐specific antibody (S235/236, Cell Signaling, Danvers, MA, USA) was used to monitor mTORC1 activation. Antibodies for neuronal nuclei (NeuN; Chemicon, Billerica, MA, USA), glial fibrillary acidic protein (GFAP; Dako), neuron‐glial antigen 2 (NG2; Millipore, Billerica, MA, USA), OX‐42 (Serotec Oxford, UK), ED1 (Serotec Oxford, UK), Ki‐67 (BD Pharmingen Oxford, UK), and myeloperoxidase (MPO; Hycult Biotech, Uden, the Netherlands) were used to evaluate neurons, astrocytes, oligodendrocyte progenitors, microglia/macrophages, activated microglia/macrophages, proliferation, and neutrophils, respectively. Primary antibodies were visualized using appropriate secondary antibodies (Cy3 or DyLight™ 488, Jackson Laboratories, Sacramento, CA, USA).
Spinal cord sections (20 μm) mounted on gelatin‐coated slides were incubated with primary antibody at 4°C for 24 h. After washing, secondary antibodies were added and sections incubated for 1.5 h at room temperature. Primary and secondary antibodies were diluted in 0.3% Triton‐X 100 in 0.1 M PBS. Slides were coated with an anti‐fade agent (Prolonged Gold® Invitrogen, Carlsbad, CA, USA) and were visualized using fluorescence microscopy (Nikon Eclipse TE3000, Surrey, UK) if only labeled with one marker or confocal microscopy (Olympus FV1000 CLSM, Hamburg, Germany) if double‐labeled.
Spinal cord sections (20 μm) mounted on gelatin‐coated slides were incubated with primary antibody at 4°C for 24 h. After washing, secondary antibodies were added and sections incubated for 1.5 h at room temperature. Primary and secondary antibodies were diluted in 0.3% Triton‐X 100 in 0.1 M PBS. Slides were coated with an anti‐fade agent (Prolonged Gold® Invitrogen, Carlsbad, CA, USA) and were visualized using fluorescence microscopy (Nikon Eclipse TE3000, Surrey, UK) if only labeled with one marker or confocal microscopy (Olympus FV1000 CLSM, Hamburg, Germany) if double‐labeled.
8
Imaging Neutrophil-Fungal Interactions in Zebrafish
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Transgenic larvae were pre-screened for fluorescence using a zoomscope (EMS3/SyCoP3; Zeiss; Plan-NeoFluor Z objective). For multi-day imaging experiments, larvae were anesthetized and mounted in a Z-wedgi device [39 , 44 (link)] where they were oriented such that the hindbrain was fully visible. Z-series images (5 μm slices) of the hindbrain were acquired on a spinning disk confocal microscope (CSU-X; Yokogawa) with a confocal scanhead on a Zeiss Observer Z.1 inverted microscope, Plan-Apochromat NA 0.8/20x objective, and a Photometrics Evolve EMCCD camera. Between imaging sessions larvae were kept in E3-MB with PTU in individual wells of 24- or 48-well plates. Neutrophil-fungal interactions were imaged using an inverted epifluorescence microscope (Nikon Eclipse TE3000) with a Nikon Plan Fluor 20x/0.50 objective, motorized stage (Ludl Electronic Products) and Prime BSI Express camera (Teledyne Photometrics). Environmental controls were set to 37°C with 5% CO2. Images were acquired every 3 min for 12 h. Imaging of A. fumigatus stained with CFW was performed using an upright Zeiss Imager.Z2 LSM 800 laser scanning confocal microscope with Airyscan detection and a Plan-Apochromat 20x /0.8 objective. A single z plane image was acquired for each hypha. Images were captured using identical laser and exposure settings for each condition.
9
Quantification of Primitive and Definitive EPC Colonies
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Freshly isolated PBMCs (1.5 × 10 5 ) were seeded into semisolid methyl cellulose-based culture medium coated 35 mm dishes (Falcon, Corning, CA, USA) and left in a humidified incubator with 5% CO2 at 37 °C until colony appearance. The number of adherent colonies was counted between days 16-18, using a gridded scoring dish (STEMCELL Tech., Vancouver, Canada) under a phase-contrast light microscope (Eclipse TE3000; Nikon, Tokyo, Japan). Primitive EPC colony-forming units (PEPC-CFUs) and definitive EPC colonyforming units (DEPC-CFUs) were separately counted as reported elsewhere 12, 13 .
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