The number of cells in the inner cell mass (ICM) and the trophectoderm (TE) of expanded and hatched blastocysts was determined using differential staining on day 7.5 post-IVF, based on previously published methods46 (link). Briefly, embryos were placed in 0.5% (w/v; 10 μL) pronase in GMOPS (Vitrolife) to remove the zona pellucida, and then washed in GMOPS-PLUS (Vitrolife) for 5 min. Embryos were subsequently incubated in 10 μL of 10 mM trinitrobenzenesulfonic acid (TNBS) in 4 mg/mL polyvinyl pyrollidone (PVP) in simple-G1 medium (Vitrolife; G1-PVP) for 20 min, washed twice in GMOPS-PLUS then transferred to 0.1 mg/mL anti-2,4 dinitrophenol-G1-PVP (10 μL) for 10 min. Following a further wash in GMOPS-PLUS, embryos were transferred to 10 μL drops of 10% v/v guinea pig serum in 25 mg/mL propidium iodide in GMOPS for 1 min, and then placed into 10 μL drops of 0.1 mg/mL bisbenzimide for 20 min. Embryos were individually mounted in 100% glycerol on a glass slide under a coverslip. Embryos were viewed under fluorescent light using a Nikon Eclipse TS100 microscope equipped with a mercury lamp (Olympus) and images of embryos were taken using the Nikon Digital Sight DS-L2 (Nikon). The number of cells in the inner cell mass (ICM) and the trophectoderm (TE) were counted using the cell-counter tool in ImageJ software (National Institute of Health).
Digital sight ds l2
Manufactured by Nikon
Sourced in Japan
The Nikon Digital Sight DS-L2 is a digital microscope camera control unit designed for use with Nikon's Digital Sight series of microscope cameras. It provides a user interface for camera control and image capture.
Automatically generated - may contain errors
Lab products found in correlation
Eclipse ts100, by Nikon (5 mentions)
Diaphot, by Nikon (5 mentions)
Rhodamine phalloidin, by Thermo Fisher Scientific (3 mentions)
Eclipse 50i, by Nikon (2 mentions)
G mops plus, by Vitrolife (1 mentions)
G mops, by Vitrolife (1 mentions)
Mercury lamp, by Olympus (1 mentions)
Mouse monoclonal anti cb, by Swant (1 mentions)
Mouse monoclonal anti cr, by Swant (1 mentions)
Mouse monoclonal anti pv, by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Lipopolysaccharides (lps), by Merck Group (1 mentions)
Ds fi1 digital camera, by Nikon (1 mentions)
Fluorescent brightener, by Merck Group (1 mentions)
Eclipse 50i microscope, by Nikon (1 mentions)
Dfc350fx, by Leica camera (1 mentions)
Glycine buffer, by Merck Group (1 mentions)
Methyl thiazolyl tetrazolium (mtt), by Merck Group (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Photo paint x6, by Corel (1 mentions)
20 protocols using digital sight ds l2
1
Quantifying Blastocyst Cell Lineages
2
Quantification of GABA, PV, CB, and CR Neurons in Spinal Cord
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For quantitative analysis of GABA-expressing neurons, serial coronal sections (7 µm) from the location of the open defect (exposed area) of the lumbar cord in the SBA chicks, and from a similar location in the normal chicks, were stained with a rabbit polyclonal anti-GABA antibody (1 : 1500; Sigma). The tissues were observed under a Nikon Eclipse E800 light microscope, and images were acquired using a charge-coupled device (CCD) camera attached to the microscope (Nikon Digital Sight DS-L2). For analysis of GABAexpressing neurons, all GABA-immunopositive cells with a rounded profile in superficial dorsal horn laminae I-III were considered and counted. The total num-ber of neurons in the superficial dorsal horn on each side of the spinal cord was calculated and averaged across six cross sections per chick at each age; each group comprised six chicks of each age.
For quantitative analysis of PV-, CB-, and CR-expressing neurons, serial coronal sections were stained with mouse monoclonal anti-PV (1 : 500; Sigma), mouse monoclonal anti-CB, or mouse monoclonal anti-CR (1 : 500; SWANT) antibody, and PV-, CB-, and CR-immunopositive cells were enumerated as described above.
For quantitative analysis of PV-, CB-, and CR-expressing neurons, serial coronal sections were stained with mouse monoclonal anti-PV (1 : 500; Sigma), mouse monoclonal anti-CB, or mouse monoclonal anti-CR (1 : 500; SWANT) antibody, and PV-, CB-, and CR-immunopositive cells were enumerated as described above.
3
Microscopy and Morphometry of Tardigrades
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Specimens for light microscopy and morphometry were mounted on microscope slides in Hoyer's medium according to Morek et al. [35 (link)] and, together with the material cited above, examined under a Nikon Eclipse 50i phase-contrast microscope (PCM) fitted with a Nikon Digital Sight DS-L2 digital camera. Specimens for imaging in SEM were prepared according to Stec et al. [26 (link)]. Bucco-pharyngeal apparatuses were extracted following the sodium hypochlorite protocol provided by Eibye-Jacobsen [36 (link)] with modifications described in Gąsiorek et al. [37 (link)]. Both animals and apparatuses were examined under high vacuum in a Versa 3D DualBeam SEM at the ATOMIN facility of Jagiellonian University, Kraków, Poland. For deep structures that could not be fully focused in a single photograph, a series of one to three images were taken every ca 0.1 µm and then assembled with Corel into a single deep-focus image.
4
Fungal Isolate Characterization Protocol
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Fungal isolates were grown at 22°C on PDA and oatmeal agar in the dark for 2 weeks before observation. Culture characteristics, including texture, density, color, growth front, transparency, and zonation, were visually examined [25] . Colony colors observed from the surface and reverse, both top and back, were described using the color chart of Rayner [26] .
Microscopic observation of morphology of fungal isolates was conducted using cultures grown on PDA and synthetic nutrient agar [27] under continuous n-UV light (400–315 nm). A Nikon Eclipse (D v4.50, Nikon, Tokyo) 80i light microscope equipped with a Digital Sight DS-L2 camera (Nikon, Tokyo) and NIS-Element software were used to capture digital images. For each isolate, at least 30 measurements were obtained for each structure. Measurements are given as minimum (lower limit of a 95% confidence interval), average, and maximum (upper limit of a 95% confidence interval). Based on morphology observation, Fusarium isolates were identified into genus level.
Microscopic observation of morphology of fungal isolates was conducted using cultures grown on PDA and synthetic nutrient agar [27] under continuous n-UV light (400–315 nm). A Nikon Eclipse (D v4.50, Nikon, Tokyo) 80i light microscope equipped with a Digital Sight DS-L2 camera (Nikon, Tokyo) and NIS-Element software were used to capture digital images. For each isolate, at least 30 measurements were obtained for each structure. Measurements are given as minimum (lower limit of a 95% confidence interval), average, and maximum (upper limit of a 95% confidence interval). Based on morphology observation, Fusarium isolates were identified into genus level.
5
Phagocytosis of Streptococcus sanguinis by LPS-activated RAW 246.7 Cells
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RAW 246.7 monocyte/macrophage-like cells were activated with lipopolysaccharide (LPS; 100 ng ml−1, Sigma) with and without diluted (1 : 1) conditioned media collected from 24 h cultures of T cells alone (T CM), PDL cells and T cells (PDL-T CM) and IFNγ-primed PDL cells and T cells (IFNγ-PDL-T CM). After 24 h in culture, cell morphology was observed under a light microscope (Nikon Eclipse TS100), and photomicrographs of osteoclast appearances were taken using a Nikon Digital sight DS-L2. The number of viable cells was determined by MTT assay, and the expression of IL-1β and TNFα genes was determined by qPCR, as described above. For a phagocytosis assay, LPS-activated RAW 246.7 cells were co-cultured with heat-killed CFSE-stained Streptococcus sanguinis, at a concentration of 10 bacteria per one RAW 246.7 cell, for 60 min to allow phagocytosis. The levels of CFSE+ and CFSE− RAW 246.7 cells were determined by FCM. Confocal fluorescence of paraformaldehyde-fixed culture stained with 0.1 nM rhodamine phalloidin (Thermo Fisher Scientific, Waltham, MA, USA) and 1 µM DAPI (Sigma) was also carried out to confirm cellular localization of CSFE-stained bacteria in RAW 246.7 cells. CSFE-stained bacteria were stained green, while nuclei and intracellular actin filaments were stained blue and red, respectively.
6
Osteoclast Differentiation from PBMCs
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PBMCs, isolated as described above, were used in this assay. Cells were induced with osteoclastic medium (OCM), consisting of 25 µg ml−1 macrophage colony-stimulating factor (MCSF) and 25 µg ml−1 receptor activator of NF-κB ligand (RANKL), in the presence of 1 : 1 diluted conditioned media collected from 24 h cultures of T CM, PDL-T CM and IFNγ-PDL-T CM, for 14 days. The presence of tartrate-resistant acid phosphatase (TRAP+) multinucleated cells was observed under a light microscope (Nikon Eclipse TS100), and photomicrographs of osteoclast appearances were taken using a Nikon Digital sight DS-L2. The number of osteoclasts (TRAP+ cells with more than two nuclei) per area (mm3) was also counted manually. The expression of osteoclast differentiation TRAP and cathepsin K (CTSK) genes was determined by qPCR, as described above.
7
Embryonic Development Phenotyping
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Embryos were regularly observed in bright field 1-10× magnification for the assessment of gross morphological phenotype. No obvious differences between sex steroid-treated and control embryos were detected regarding speed of development or deformities, except for changes in pigmentation, gill branches and blood vessels. To assess these changes, embryos at st 40 and 45 were anesthetized (as described above) for static imaging of embryos alive (Digital Sight DS-L2, Nikon), the resulting pictures were further analyzed.
8
Evaluating Cell Viability and Morphology
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Cells were cultured at a density of 3 × 103 cells well−1 in 96-well plates for 18 h. Cells were treated as indicated in each experiment. After indicated time in culture, the cells were fixed with 4% paraformaldehyde (PFA) for 15 min and stained with 0.05% w/v crystal violet solution for 15 min. The cell morphology was examined under a light microscope (Nikon Eclipse TS100), and photomicrographs of cell appearances were taken using a Nikon Digital sight DS-L2.
For cell viability assay, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to examine the viability of MSCs. After being cultured at the indicated times, the cells were incubated with 0.2% MTT solution for 4 h at 37°C, and the reaction was then stopped by adding dimethyl sulfoxide (DMSO) and glycine buffer. The end product colour was subsequently analysed by measuring an absorbance at 490 nm (A490) which corresponds to the viability of cells. Cell viability is expressed as the mean percentage of control (100%). Data are presented as the mean percentage ± s.d. from three independent experiments.
For cell viability assay, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to examine the viability of MSCs. After being cultured at the indicated times, the cells were incubated with 0.2% MTT solution for 4 h at 37°C, and the reaction was then stopped by adding dimethyl sulfoxide (DMSO) and glycine buffer. The end product colour was subsequently analysed by measuring an absorbance at 490 nm (A490) which corresponds to the viability of cells. Cell viability is expressed as the mean percentage of control (100%). Data are presented as the mean percentage ± s.d. from three independent experiments.
9
Microscopic Imaging of Fungal Cell Wall
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Images of cells after 24 h of growth in the presence of caspofungin (Figure 1 ) were directly obtained from the microcultures used for the susceptibility assays with a Nikon Eclipse 50i microscope, a Nikon Plan FLUOR 20×/0.45 objective, a Nikon Ds-Fi1 digital camera, and a Nikon Digital Sight DS-L2 control unit, as previously described [50 (link)]. For cell wall staining and GFP-GS localization, early log-phase cells with or without GFP-labelled GS proteins were grown at 28 °C in YES liquid medium. Before imaging, the cells were concentrated (1000× g, 1 min) and resuspended by adding a solution of Calcofluor White (CW; 50 μg/mL final concentration; Fluorescent Brightener 28 Sigma-Aldrich, Burlington, MA, USA) to the sample and visualized using the appropriate filters.
Images were obtained with a Leica DM RXA fluorescence microscope, a PL APO 63×/1.32 oil PH3 objective, a digital camera (DFC350FX; Leica, Wetzlar, Germany), and CW4000 cytoFISH software (Leica). Images were processed with the Adobe Photoshop software. All the analyses were repeated in three independent experiments and representative images of the analyzed phenotypes were indistinctly selected from the experiments.
Images were obtained with a Leica DM RXA fluorescence microscope, a PL APO 63×/1.32 oil PH3 objective, a digital camera (DFC350FX; Leica, Wetzlar, Germany), and CW4000 cytoFISH software (Leica). Images were processed with the Adobe Photoshop software. All the analyses were repeated in three independent experiments and representative images of the analyzed phenotypes were indistinctly selected from the experiments.
10
GUS Staining of Arabidopsis Seedlings
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GUS staining was performed as described earlier (Okamoto et al., 2008) . In brief, 3-day-old seedlings were transferred to 1.5 mM cesium chloridecontaining agar plates and grown vertically at 23 C under continuous white light. After 3 days incubation, seedlings were transferred to GUS staining buffer (100 mM sodium phosphate [pH 7.0], 10 mM EDTA, 0.5 mM potassium ferricyanide, 0.5 mM potassium ferrocyanide, and 0.1% Triton X-100) containing 1 mM X-gluc and incubated at 37 C in the dark for 3 h. The roots were imaged with a light microscope (Nikon Diaphot) equipped with a digital camera control unit (Digital Sight [DS-L2]; Nikon, Japan).
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