Sirius red, RNAscope and H&E stainings were captured with a Nikon Ti2 Eclipse microscope, equipped with a Nikon Digital Sight D5-VI1 color camera. O-R-O stainings and IHC stainings were taken using a Nikon Eclipse Ci microscope, with a color camera or a S360 Hamamatsu Slidescanner (CECAD Imaging Facility, Cologne). Color brightfield images of HE staining for the spatial transcriptomics experiment were captured with a Nikon Ti2 Eclipse microscope, equipped with a Nikon Digital Sight D5-VI1 color camera and using a 10×/ 0.3 NA CFI Plan-Flour objective. Tilescan image acquisition was carried out using the NIS Elements software.
Eclipse ti2 microscope
Manufactured by Nikon
Sourced in Japan, United States, Germany, United Kingdom
The Eclipse Ti2 is a high-performance inverted research microscope designed for advanced microscopy applications. It features a stable, vibration-resistant optical system and a wide range of interchangeable accessories to accommodate various imaging techniques, including brightfield, phase contrast, and fluorescence microscopy.
Automatically generated - may contain errors
Lab products found in correlation
Hoechst 33342, by Thermo Fisher Scientific (12 mentions)
Nis elements software, by Nikon (9 mentions)
Nis element, by Nikon (8 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (8 mentions)
Ds qi2 camera, by Nikon (5 mentions)
Transwell inserts in 24 well dishes, by Corning (5 mentions)
Alexa fluor 568, by Thermo Fisher Scientific (4 mentions)
Fura 2 am, by Thermo Fisher Scientific (4 mentions)
Csu w1, by Yokogawa (3 mentions)
Goat serum, by Thermo Fisher Scientific (3 mentions)
Cfi plan apo lambda 100x oil objective, by Nikon (3 mentions)
Thorlab camera dcc154m gl, by Thorlabs (3 mentions)
A1r confocal scanhead, by Nikon (3 mentions)
Elements analysis 5, by Nikon (3 mentions)
Nis elements advanced research software, by Nikon (3 mentions)
Ds qi2, by Nikon (3 mentions)
Prime bsi camera, by Cairn Research (3 mentions)
Optospin filter wheel, by Cairn Research (3 mentions)
Vt isim super resolution confocal scan head, by Visitech (3 mentions)
Prolong diamond antifade mountant, by Thermo Fisher Scientific (3 mentions)
261 protocols using eclipse ti2 microscope
1
Multimodal Tissue Imaging Protocols
2
Live Cell Imaging of Raf1-RBD-GFP
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For live cell imaging of cells in a rAprA gradient, cells were resuspended to 1 × 105 cells/ml and exposed to a gradient in the well of an eight-well slide for 30 min as described above. Differential interference contrast (DIC) and fluorescence images of live cells were taken with a 20× objective on a Nikon Ti2 Eclipse microscope. For live cell imaging of cells in a uniform concentration of rAprA, the cells were prepared as above. rAprA in 20 mM NaPO4, pH 7.4, was added to a final concentration of 300 ng/ml, and this was mixed gently and then allowed to incubate for 30 min. Controls were treated similarly, adding an equal concentration of 20 mM NaPO4, pH 7.4. For some experiments, the medium was gently removed after 1 h and 300 μl of fresh HL5 was added to each well. Images were taken with a 40× objective on a Nikon Ti2 Eclipse microscope. FIJI ImageJ software (Schindelin et al., 2012 (link)) was used to analyze and quantify fluorescence. Quantification was done as previously described (Mouneimne et al., 2006 (link)) with the exception that the ratio was ([integrated Raf1-RBD-GFP intensity in the cortical region at the front and sides of the cell]/3) divided by (integrated Raf1-RBD-GFP intensity in the cortical region at the back of the cell).
3
Senescence-Associated β-Galactosidase Assay
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The expression of SA-β-Gal in MRC-5 cells and mouse kidney cells was detected by an SA-β-Gal staining kit. The cells were seeded in 24-well plates (10000 cells per well) in a final volume of 1 mL. After 72 h of administration, the cells were washed three times with PBS, fixed at room temperature for 15 min, and stained overnight with fresh staining solution. In the next day, the cells were washed three times with PBS, fixed at room temperature, stained with DAPI, and analyzed and photographed with a Nikon Eclipse Ti2 microscope. The experiments were independently repeated three times. Mouse kidneys were embedded in optimal cutting temperature (OCT) compound, frozen over dry ice, and then cut into 20 μm sections using a freezing sliding microtome. The staining steps were consistent with those used above for MRC-5 cells. Three sections per group were photographed using a Nikon Eclipse Ti2 microscope.
4
Adipose Tissue Macrophage Imaging
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6-wk-old C3(1)-TAg mice were fed an LFD or HFD for 6 wk. Mammary adipose tissue was isolated at 12 wk of age, a time point that coincides with DCIS (Holzer et al., 2003 (link)). Adipose tissue was sectioned (5 µm), stained with anti-F4/80 antibody (Abcam) to visualize ATMs, and counterstained with hematoxylin. Images were acquired with a Nikon Eclipse Ti2 microscope with the following setting: brightfield, objective magnification 20 and objective numerical aperture 0.45, room temperature, Color Camera Nikon DS-Ri2, and NIS-Element Version 5.02 software.
Mammary and visceral fat was obtained from WT and mNox2−/− mice fed a LFD or HFD for 10 wk. Adipose tissue was sectioned (5 µm) and stained with antibodies against MAC2 (Cedarlane) and PLIN2 (Abcam). Fluorescence images were acquired with a Nikon Eclipse Ti2 microscope with the following setting: objective magnification 20, objective numerical aperture 0.45, room temperature, emission wavelength of DAPI (457.5 nm), GFP (535.0 nm), and RFP (610 nm), Camera Nikon DS-Qi2, and NIS-Element Version 5.02 software.
Mammary and visceral fat was obtained from WT and mNox2−/− mice fed a LFD or HFD for 10 wk. Adipose tissue was sectioned (5 µm) and stained with antibodies against MAC2 (Cedarlane) and PLIN2 (Abcam). Fluorescence images were acquired with a Nikon Eclipse Ti2 microscope with the following setting: objective magnification 20, objective numerical aperture 0.45, room temperature, emission wavelength of DAPI (457.5 nm), GFP (535.0 nm), and RFP (610 nm), Camera Nikon DS-Qi2, and NIS-Element Version 5.02 software.
5
Epithelial-Mesenchymal Transition Dynamics
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Cells were grown and treated with 5 ng·mL−1 TGFβ1 on a glass‐bottom dish, and EMT time‐lapse imaging (Movie S1 ) was performed with an TCS SP8 microscope (Leica Microsystems, Wetzlar, Germany). The time‐lapse images were acquired every 15 min for a total imaging time of 48 h. After induction of EMT by TGFβ1 for 7 days, the cells were grown without TGFβ1 and MET imaging was performed with an ECLIPSE Ti2 microscope (Nikon, Minato, Japan). Images were acquired every 30 min with a total imaging time of 62 h (Movie S2 ). Five days after mammosphere formation in ultra‐low attachment condition (3D), mammospheres were grown with (complete EMT) or without (partial EMT) 5 ng·mL−1 TGFβ1 on a glass‐bottom dish, and time‐lapse imaging was performed with an ECLIPSE Ti2 microscope (Nikon, Minato, Japan). Images were acquired every 8 min for a total imaging time of 55 h (Movie S3 ).
For RFP intensity quantification of migratory cells from mammospheres, 3 cells were selected at random from each quadrant of one sphere in the differential interference contrast (DIC) channel (total 60 cells per condition). Then, the mean RFP intensity was measured in the RFP channel byimage j software. All experiments were recorded using identical image acquisition conditions.
For RFP intensity quantification of migratory cells from mammospheres, 3 cells were selected at random from each quadrant of one sphere in the differential interference contrast (DIC) channel (total 60 cells per condition). Then, the mean RFP intensity was measured in the RFP channel by
6
Quantifying Decellularization Efficiency
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The decellularization efficiency of the qualitatively selected optimized protocol was quantified. Regions of Interest (ROI) were defined via manual segmentation of fascicles using the NIS-elements software. All subsequent steps were based on the ROI. Autofluorescence of the tissue was subtracted using non-stained negative controls. The remaining MBP, β-tubulin III and DAPI signal after decellularization were then plotted in relation to the signal of unprocessed tissue segments. Images were taken using a Nikon Ti2 Eclipse microscope (Nikon, Chiyoda, Tokio, Japan) and CFI Plan Apo Lambda objectives. Fluorescence images were acquired by a photometrics Prime95B camera using a 5-bandpass light filter. Color images were acquired by a Nikon DS-Ri2 camera.
7
Imaging of Non-Adherent Hematopoietic Cells
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To avoid Z-focal plane drop-out in solid media, non-adherent hematopoietic 32D, TF-1, or HEL cells were seeded in liquid medium in 8- or 18-well chambers (Ibidi, Gräfelfing, Germany), allowed to sediment for 30 min, and imaged in multi-position mode every 1.5–2 min for more than 15 h. Recording was performed with a 20 × 0.75NA objective using the fluorescence-widefield mode of an X-light spinning disk (CrestOptics, Roma, Italy) mounted on a Nikon Ti2 Eclipse microscope (Nikon, Melville, NY, U.S.A.) with the environmental control system UNO-T-H-CO2 (Okölab, Ottaviano, NA, Italy). Samples were illuminated with a LED light engine SpectraX (Lumecor, Beaverton, OR, USA) at very low intensities (< 3% of line source power and < 1 s acquisition per channel), through mCherry filter sets for chromatin imaging (mCherry), and GFP for assessing single cell expression of the EV-, CALRdel52- or JAK2V617F.
8
Time-lapse Imaging of Spheroid Cultures
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Multichamber LabTek slides (Nunc) were used for imaging. Spheroids were placed in individual agarose wells (ca. 1 mm in diameter) within the chambers to retain them in the field of view. The wells were created by coating the bottom of the chambers with a 2- to 3-mm-thick layer of 1% agarose in HM and puncturing holes using a 1-ml micropipette tip after the gel solidified. The chamber was then filled with the desired imaging medium. In case of sucrose treatments, the agarose gel contained sucrose in concentrations equal to the overlaying medium. For inhibitor treatments, drug concentration in the imaging medium was adjusted, taking the volume of agarose into account. To prevent diluting the imaging medium in the chamber, closed spheroids were first transferred to a dish with bigger volume (ca. 10 ml) of the imaging medium and positioned in the imaging wells only after this wash. Samples were imaged every 10 min at 21°C under bright-field illumination using the Nikon Ti2-Eclipse microscope with a 10× CFI Plan Apochromat Lambda objective (Nikon) and iXon Ultra 888 electron-multiplying charge-coupled device (EMCCD) camera (Andor). Images were acquired in the 1024 × 1024 format, with pixel size of 1.3 μm.
9
Quantifying SEC31A Puncta in 4-PBA-Treated HEK-293 Cells
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HEK-293 TREx cells were seeded on coverslips in 6-well plates and then treated with or without 10 mM 4-PBA for 4 h. Then, cells were fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.1% Saponin for 10 min, blocked with 1% bovine serum albumin for 1 h, and stained with 1:100 anti-SEC31A (BD Biosciences, #612350) overnight at 4 °C. The next day, coverslips were stained with anti-mouse 1:250 Alexa Fluor 568 (Thermo Fisher Scientific, #A11031) for 1 h at room temperature. Coverslips were washed 3 × 3-min with phosphate-buffered saline after fixing, primary and secondary antibody incubations; cells were stained with 10 μg/μL DAPI (Merck) for 15 min. Coverslips were mounted in Prolong Diamond Antifade (Thermo Fisher Scientific). Z-stack images at 0.9-μm intervals were taken on a Nikon Ti2 Eclipse microscope with a 60x/1.4 numerical aperture oil immersion objective and an Orca-flash 4.0 camera (Hamatsu). Images were collected using NIS Elements AR 5.11.03 software, followed by processing in Fiji. SEC31A puncta were quantified from merged Z-stack images using an in-house Fiji macro (41 (link)).
10
Time-Lapse Imaging of Antibiotic-Treated Bacteria
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Bacterial cultures (OD600 = 0.01) were prepared with or without antibiotic treatment in iron-limited
MHB broth as mentioned above. For time-lapse imaging, the 96-well
plate was incubated at 37 °C. Image acquisition was carried out
with 30 min intervals over 24 h. The time-lapse images were collected
on a Nikon-Ti2 Eclipse microscope at differential interference contrast
view equipped with a 100× oil-immersion objective lens.
MHB broth as mentioned above. For time-lapse imaging, the 96-well
plate was incubated at 37 °C. Image acquisition was carried out
with 30 min intervals over 24 h. The time-lapse images were collected
on a Nikon-Ti2 Eclipse microscope at differential interference contrast
view equipped with a 100× oil-immersion objective lens.
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