Cells grown on coverslips were preextracted in PHEM buffer (60 mM PIPES, 25 mM HEPES, 10 mM EGTA [ethylene glycol tetraacetic acid], 4 mM MgSO4, pH 7.0) containing 0.05% digitonin for 5 min and fixed by 4% paraformaldehyde in PBS at RT for 10 min. Cells were then permeabilized with PBST (phosphate-buffered saline containing 0.1% Triton X-100) and blocked with PBST containing 3% bovine serum albumin for 30 min. Primary antibodies were incubated at RT for 2 h and secondary antibodies for 1 h. DNA was stained by Hoechest 33342 (1 μg/ml) for 1 min. Cell images were acquired either by a 20× objective lens in a Nikon Eclipse Ti-E microscope for statistics and by a 60× objective lens in a Zeiss confocal microscope or a 100× objective lens in a Nikon Eclipse Ti-E microscope for representative images. The average of total protein intensity in small areas of two centrosomes subtracted by similar areas of mitotic cytoplasm in the same cell was used for quantification statistics. Law images from Nikon microscope were deconvoluted by NIS-element (Nikon) software and processed with ImageJ and Photoshop (Adobe). Fluorescence time-lapse imaging of A549 cells expressing CFP-H2B was recorded every 10 min for a total duration of 24 h with a 10× objective in a Nikon Eclipse Ti-E microscope equipped with a temperature- and CO2-controlled stage incubation unit (Okolab).
Eclipse ti e microscope
Manufactured by Nikon
Sourced in Japan, United States, United Kingdom, Germany, Netherlands
The Eclipse Ti-E microscope is a research-grade inverted microscope designed for advanced live-cell imaging and high-resolution microscopy. It features a motorized focus drive, fluorescence illumination, and compatibility with a variety of imaging techniques, including phase contrast, DIC, and fluorescence. The Eclipse Ti-E provides a stable and versatile platform for a wide range of biological and materials science applications.
Automatically generated - may contain errors
Lab products found in correlation
Nis elements software, by Nikon (13 mentions)
Nis element, by Nikon (8 mentions)
Lsm 780, by Zeiss (8 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (8 mentions)
Perfect focus system, by Nikon (7 mentions)
Prolong gold, by Thermo Fisher Scientific (6 mentions)
Triton x 100, by Merck Group (6 mentions)
Ixon ultra du 897 emccd camera, by Oxford Instruments (6 mentions)
Poly l lysine (pll), by Merck Group (5 mentions)
Flash4 v2 cmos camera, by Hamamatsu Photonics (5 mentions)
A1 confocal module, by Nikon (5 mentions)
Stage top cage incubator, by Okolab (5 mentions)
Matrigel, by BD (5 mentions)
Fura 2 am, by Thermo Fisher Scientific (5 mentions)
Hoechst 33342, by Thermo Fisher Scientific (5 mentions)
Nis elements ar software, by Nikon (5 mentions)
Metamorph software, by Molecular Devices (4 mentions)
Evolve emccd camera, by Nikon (4 mentions)
Dulbecco modified eagle medium (dmem), by Merck Group (4 mentions)
Glutamax, by Thermo Fisher Scientific (4 mentions)
321 protocols using eclipse ti e microscope
1
Imaging of Centrosome Protein Localization
2
Visualization and Quantification of STIM1β in Cells and Tissues
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HEK293, HeLa, and U87 MG cells were grown on 35‐mm glass‐bottom dishes. After 24 h, cells were fixed in 4% paraformaldehyde (PFA) solution in PBS at room temperature for 15 min and then permeabilized at room temperature with 0.1% Triton X‐100/PBS solution, and subsequently blocked with 3% BSA for 60 min. For the overexpression of target proteins, cells were fixed 16 h after transfection. The dish was incubated with anti‐STIMβ antibody diluted 1:200 (endogenous) or 1:1000 (overexpression) overnight at 4 °C. Cells were then washed three times with PBS and incubated with Donkey anti‐Goat Secondary Antibody, Alexa Fluor 488 or 568 with 1:1000 dilution for 1 h. The nucleus was stained with DAPI for 2 min. The cells were then captured using a Nikon Eclipse Ti‐E microscope and analyzed with the Nikon NIS‐Elements AR package software.
The STIM1β levels in brain tissue were evaluated by IHC using anti‐STIM1β antibody on a purchased human brain cancer tissue array (GL805a, US Biomax Inc.). The tissue array slide was deparaffinized and stained with an anti‐STIM1β antibody. IHC‐stained tissue images were obtained and evaluated at an inverted Nikon Eclipse Ti‐E microscope with 40× oil lens. The relative expression of STIM1β was normalized against the sample with the highest immunostaining intensity.
The STIM1β levels in brain tissue were evaluated by IHC using anti‐STIM1β antibody on a purchased human brain cancer tissue array (GL805a, US Biomax Inc.). The tissue array slide was deparaffinized and stained with an anti‐STIM1β antibody. IHC‐stained tissue images were obtained and evaluated at an inverted Nikon Eclipse Ti‐E microscope with 40× oil lens. The relative expression of STIM1β was normalized against the sample with the highest immunostaining intensity.
3
Transwell-based Cell Migration Assay
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HMEC (10 × 104 cells/well) and PTEC (5 × 104 cells/well) were plated in 24-well plates (2 wells/condition) using EndoGRO MV-VEGF medium. After 24 h, Transwell® permeable supports (6.5 mm inserts with an 8 µm pore polycarbonate membrane) were equilibrated for 20 s at 37 °C using RPMI 0% FBS medium. The equilibrated Transwell® inserts were then placed over the wells containing the previously plated HMEC and PTEC. PC3 cells (5 × 104 cells/insert) were seeded in the Transwell® inserts using RPMI 0% FBS and incubated for 24 h. Transwell® inserts were then washed in PBS twice and fixed in methanol for 30’. PC3 cells were then colored with 0.5% Crystal Violet in methanol for 20 s at room temperature and after washed in PBS. PC3 cells that did not migrate thought the membrane pores were removed from the upper side of the membrane using a cotton bud. PC3 cells that migrated thought the membrane pores were then counted using an Eclipse Ti-E Nikon microscope with a 10×/0.25 NA Plan objective.
4
Multicolor TIRF Microscopy Imaging
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In vitro assays using fluorescence imaging with no force application were carried out with an inverted Eclipse Ti-E Nikon microscope equipped with a 1.49 numerical aperture 100× oil objective, Perfect Focus system, and Andor iXon3 CCD camera (Cambridge Scientific, MA, USA), as in (75 (link)). Three diode lasers (488, 561, and 640 nm from Coherent, CA, USA) were used as a light source with a C-TIRF Quad cube (Chroma, VT, USA). The multicolor TIRF was implemented by rapidly switching between different laser wavelengths with an acousto-optical tunable filter and corresponding emission filter wheels. Under these conditions, the microscope produced 512 × 512 pixel images with a resolution of 0.14 μm/pixel in both the x and y directions. Illumination was carried out with a partially opened iris diaphragm to limit laser illumination only by the field of view during each exposure. Each imaging channel was recorded with a 300-ms exposure time unless otherwise specified. All experiments were carried out at 32°C by heating the objective with an objective heater (Bioptechs, PA, USA).
5
Cell Migration Assay for Cancer Research
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Commercially available Chemicon QCM Chemotaxis 5 mum 24-well migration assay system (CHEMICON, Millipore, USA) was used to access the cell migratory ability [19 (link)]. Serum starved HGC-27 and NCI-N87 cells were plated at 2 × 104 cells/well seeding densities on the upper layer of the inserts. Chemo-attractive medium (RPMI 1640 medium with 10% FBS) was placed below the cell-permeable membrane in the lower chambers. After 24 hours of incubation, cells were washed in phosphate buffer saline (PBS) and the cells at the bottom of the chamber were carefully wiped, followed by fixation and staining were followed respectively. The cells were counted using an Eclipse Ti-E Nikon microscope (Nanjing, China).
6
Spinning Disc Confocal Microscopy Protocol
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Imaging was performed using an inverted Nikon Eclipse Ti-E microscope with perfect focus system, an oil immersion objective (Nikon Plan Apo λ 100× NA 1.45), using two EMCCD cameras (Photometrics Evolve 512), which are mounted on a spinning disc unit (CSU-W1, Yokogawa). TIRF illumination was generated with the FRAP/TIRF system Ilas2 (Roper Scientific). A custom-made objective heater was used for temperature control of the samples. The imaging software used was Metamorph 7.8.8.0 with system specific routines (Ilas2) for streaming, time lapse and scanning slide acquisition.
7
DAPT-Loaded MnO2 Nanosheets' Effect on Neurogenesis
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To study the effect of DAPT-loaded MnO2 nanosheets on neurogenesis of SMART spheroids, we first loaded varying concentrations of DAPT on the surface of the MnO2 nanosheets at concentrations from 0 to 50 μM as previously described. These drug-loaded nanosheets were then used to form spheroids, and then cells were transferred to 24-well plates and allowed to differentiate for 7 days by bFGF withdrawal. Cells were then fixed with formalin or lysed with TRIzol, as previously described. Cells that were fixed were stained and imaged for a nuclear marker (Hoechst) and a neuronal marker (TuJ1). The cells were then imaged using the Nikon Eclipse Ti-E microscope, and all images were analyzed for percent differentiation using the ImageJ software, where nuclei were counted and cells were studied for whether they were TuJ1 positive. Besides, the axon length was studied using the NeuronJ software in ImageJ for axon tracing using the TuJ1 signal. For qRT-PCR experiments, the TRIzol samples were collected and reverse-transcribed using the SuperScript III First-Strand Synthesis System (Life Technologies). The cDNA was then used for qPCR using a StepOnePlus RT-PCR system (Applied Biosystems), a Power SYBR Green PCR master mix (Applied Biosystems), and primers specific to each target gene (TuJ1 and GFAP). All samples were normalized to GAPDH gene expression.
8
TIRF Microscopy and ICS Imaging of PANX1
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TIRF microscopy images were acquired on an inverted Nikon Eclipse Ti-E microscope, equipped with a 100 × 1.49 NA objective (Nikon), an iXon EMCCD camera (Andor), laser lines at 405, 488, 561, and 647 nm (Agilent), a multiband pass ZET405/488/561/647× excitation filter (Chroma), a quad-band ZT405/488/561/647 dichroic mirror (Chroma), and appropriate emission filters for imaging of mRFP (600/50 nm, Chroma) and GFP (525/50 nm, Chroma). Illumination was performed by TIRF to ensure exclusive illumination of the plasma membrane.
For ICS, the cells are fixed with 100% methanol at −20°C for 10 min. After washing with PBS, they were permeabilized with 1% Triton X-100 at 37°C for 30 min. After blocking, add primary C-terminus anti-PANX1 (Santa Cruz Biotech.) or N-terminus anti-PANX1 (Alomone Labs) was added and incubated at 4°C at 12 h followed by addition of second anti-mouse-PE (Santa Cruz) at room temperature for 30 min. After washing, the cover slips containing cells were mounted and observed using a confocal microscope (Leica).
For ICS, the cells are fixed with 100% methanol at −20°C for 10 min. After washing with PBS, they were permeabilized with 1% Triton X-100 at 37°C for 30 min. After blocking, add primary C-terminus anti-PANX1 (Santa Cruz Biotech.) or N-terminus anti-PANX1 (Alomone Labs) was added and incubated at 4°C at 12 h followed by addition of second anti-mouse-PE (Santa Cruz) at room temperature for 30 min. After washing, the cover slips containing cells were mounted and observed using a confocal microscope (Leica).
9
Multimodal Microscopy Imaging Protocol
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Confocal microscopy images were acquired on a Zeiss LSM 700 confocal laser scanning microscope equipped with a C-Apochromat 40×/1.2 W Korr objective, excitation lasers at 405 nm, 488 nm, 555 nm, and 639 nm, a multiband 405/488/555/639 beam splitter and appropriate emission filters for the detection of DAPI, Alexa Fluor 488, Cy3B, Rhodamine and Alexa Fluor 647. Images were acquired in 12-bit mode and the same settings were used across all samples. Confocal z-stacks were collected over the entire thickness of each cell in 0.34-μm slice intervals.
Wide field microscopy images were acquired on a Nikon Eclipse Ti-E microscope equipped with a 40× objective (Nikon), an X-Cite 120XL fluorescence illumination system, an iXon Ultra EMCCD camera (Andor) and appropriate filter sets for DAPI (Ex: 430DF24, Dich.:458DiO2,Em: 483DF32, Semrock) and A647 (Ex: 628DF40, Dich.:660DiO2,Em: 692DF40, Semrock) detections.
Wide field microscopy images were acquired on a Nikon Eclipse Ti-E microscope equipped with a 40× objective (Nikon), an X-Cite 120XL fluorescence illumination system, an iXon Ultra EMCCD camera (Andor) and appropriate filter sets for DAPI (Ex: 430DF24, Dich.:458DiO2,Em: 483DF32, Semrock) and A647 (Ex: 628DF40, Dich.:660DiO2,Em: 692DF40, Semrock) detections.
10
Live-cell Imaging of Cellular Dynamics
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Cells (15,000 cells/well) were plated on 12 well glass bottom plates (CellVis) which were coated with poly L-lysine (Sigma) and allowed to attach for 48 h. The cells were grown in the appropriate media as mentioned above and then rinsed with PBS and given DMEM Fluorobrite (ThermoFisher) media with 2% FBS,100 units/mL penicillin, 100 μg/mL streptomycin, 25 ng/mL amphotericin B, and 1x Glutamax (ThermoFisher). Cells were imaged every 15–20 min for 24–48 h by a Nikon Eclipse Ti-E microscope. Data was acquired using the NIS Elements AR 5.21.02 software. A thin layer of mineral oil was used to prevent evaporation. Temperature (37 °C) and 5% CO2 levels were maintained using the OKO labs incubation system. H2B-CFP was imaged using the C-FL AT ECFP/Cerulean Filter Set (Chroma) for 20–40 ms. FOXO1–mVenus was imaged using (Chroma) ET-EYFP Filter Set for 600–800 ms, p53-mCherry was imaged using (Chroma) AT-TR/mCH Filter Set for 600–800 ms. Movies were analyzed using p53Cinema73 (link).
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