The Matrigel tube formation assay was also performed to assess in vitro angiogenesis (Arutyunyan et al., 2016 (link)). Growth factor-reduced Matrigel (BD) was placed in 96-well culture plates (50 μl per well) and allowed to rest at 37°C for 40 min. Then, 3 × 104 EA.hy926 endothelial cells were added to each well and incubated in basic medium with various concentrations of leptin for 9 h. Endothelial cells forming capillary-like tubes were photographed with an inverted microscope (Nikon Eclipse 80i; Nikon, Tokyo, Japan) and an imaging software NIS-Elements D 4.50 (Nikon Instruments, Tokyo, Japan). Five fields were counted for each well. The length of the tube was measured by ImageJ software (NIH).
Nis elements d 4
Manufactured by Nikon
Sourced in Japan, Netherlands
NIS-Elements D 4.50 is an imaging software suite developed by Nikon. It provides a platform for acquiring, analyzing, and managing digital images obtained from Nikon microscopes and cameras.
Automatically generated - may contain errors
Lab products found in correlation
Eclipse 80i, by Nikon (3 mentions)
Rabbit anti laminin antibody, by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Transwell apparatus, by Corning (1 mentions)
Ds vi1 camera, by Nikon (1 mentions)
Smz745t microscope, by Nikon (1 mentions)
Eclipse 50i, by Nikon (1 mentions)
Eclipse 50i microscopy, by Nikon (1 mentions)
Uas flp cyo, by Nikon (1 mentions)
Ubi mrfpnls, by Nikon (1 mentions)
Smz18 stereomicroscope, by Nikon (1 mentions)
12 protocols using nis elements d 4
1
Matrigel Tube Formation Assay for Angiogenesis
2
Immunofluorescence Staining of Laminin and CD31
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After the antigen retrieval step using 1 mmol/L EDTA (pH 8.0), 4-μm-thick paraffin sections were incubated with the primary antibody for laminin and CD31 staining. Rabbit anti-laminin antibody (1:30 dilution; Sigma-Aldrich, St. Louis, MO, USA) and mouse anti-CD31 antibody (1:100 dilution; CST, Boston, USA) were diluted in blocking serum at 4°C overnight, washed with PBS three times, and then treated with Alexa 488-conjugated goat anti-rabbit antibody or Alexa 647-conjugated goad anti-mouse antibody (1:500 dilution; Abcam, Cambridge, MA, USA). Then, all sections were followed by DAPI (Invitrogen, Carlsbad, CA, USA) for nuclear staining. All immunofluorescence images were captured by a fluorescence microscope (Nikon Eclipse 80i; Nikon, Tokyo, Japan) and NIS-Elements D 4.50 (Nikon Instruments, Tokyo, Japan). The numbers of positive cells were counted in three randomly selected images from grafts were averaged and are presented as means ± SD. The blood vessels quantification was analyzed with Image J software practicing the “analyze particle”.
3
Cell Migration Assay with Matrigel
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OS cells (1×105) were seeded on a polycarbonate membrane insert with or without Matrigel coating (6.5 mm in diameter with 8.0 μm pores) in a Transwell apparatus (Costar, Cambridge, MA). DMEM containing 10% FBS was added to the lower chamber, while DMEM containing 0.2% BSA was added to the upper chamber. After incubation for 12 h at 37°C, the insert was washed with PBS, and cells on the top surface of the insert were removed by wiping with a cotton swab. Cells that migrated to the bottom surface of the insert were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet. Cells were counted by using a microscope (Nikon Eclipse 80i; Nikon, Tokyo, Japan) and NIS-Elements D 4.50 (Nikon Instruments, Tokyo, Japan) at 200× magnification.
4
Intestine Imaging in il10 Mutants
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The intestine of il10e46/e46 mutants and WT zebrafish was imaged with Nikon SMZ745T microscope and DS-Vi1 camera (Nikon, Minato, Tokyo, Japan). The scale bar was measured with NIS-Elements D 4.2 software (Nikon).
5
Epidermal Cell Characterization in Plants
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Surface sterilized seeds of the indicated genotypes were cold treated and sown in Petri dishes containing half strength Murashige and Skoog (0.5 x MS) nutrient solution mix [20] , 0.5% sucrose and 0.6% agarose, or in pots containing planting mix as previously described [21] 2.5.2. Epidermal cell size and number: The 3rd and 4th leaves of 5-weekold plants were painted with transparent nail polish. After drying, the painted layer was peeled off, and photographed using a light microscope. The number of stomata and pavement cells was determined using the cell counter plugin of ImageJ software as were the area of cotyledons and leaf epidermal cells. Stomatal and pavement cell density (number in mm 2 ) was calculated. Stomatal index [Stomata * 100/(Stomata + pavement cells)] was calculated according to Royer [22] . 2.5.3. Stomatal aperture area: 3rd and 4th leaves of 4-week-old plants were treated with stomata-opening buffer as previously described [23] . The lower epidermis was peeled off and the stomatal aperture area measured using the NIS-Elements D 4.2 software (Nikon, Japan).
6
Immunofluorescence Characterization of hObs Cells
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After 24 h of serum starvation, hObs were treated for 3, 6 and 24 h with BET (10 mM). hObs cells, fixed and permeabilized as previously described [28 (link)], were blocked with PBS containing 1% bovine serum albumin. Cells were then immunostained with specific antibodies FITC or rhodamine conjugated and nuclei-revealed with DAPI staining. Slides were mounted with Moviol. Cells were observed using Nikon Eclipse 50I microscopy and images were captured using Nis-Elements D 4.00 software (Nikon Instruments Europe BV, Netherlands). Data were displayed and analyzed using Adobe Photoshop CS4. Automated quantification on immunofluorescence signal was performed by using Image J program (http://imagej.nih.gov/ij/ ) [29 ].
7
Immunostaining of C2C12 Cells
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C2C12 cells, fixed and permeabilized as described in [19 (link)], were blocked with PBS containing 1% bovine serum albumin. Slides or cells were then immunostained with specific antibodies rhodamine-conjugated and nuclei-revealed with DAPI staining. Slides were mounted with Moviol. Cells were observed using Nikon Eclipse 50I microscopy and images were captured using Nis-Elements D 4.00 software (Nikon Instruments Europe BV, Netherlands). Data were displayed and analyzed using Adobe Photoshop CS4. Live C2C12 cells were examined and images acquired by phase contrast microscopy using the same microscope and digital system described above.
8
Multiparameter Analysis of Mitochondrial Function and Oxidative Stress in C2C12 Myoblasts
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C2C12 cells were fixed and permeabilized as described [18 (link)], were blocked with PBS containing 1 % bovine serum albumin. Slides or cells were then immuno-stained with specific Rhodamine- or FITC-conjugated antibodies and nuclei were revealed with DAPI staining.
The MITO CytoPainter mitochondrial indicator is a hydrophobic compound that easily permeates intact live cells and becomes trapped in mitochondria after it enters the cells. This staining was performed on C2C12 live cells during the proliferative phase and all the differentiation phases.
Cell ROX® Oxidative Stress Reagents are fluorogenic probes designed to reliably measure ROS in live cells. The cell-permeable reagents are non-fluorescent or very faintly fluorescent while in a reduced state, and during oxidation exhibit a strong fluorogenic signal. Cell ROX® Orange Reagents are localized in the cytoplasm. This staining was performed on C2C12 live cells during the proliferative phase.
Slides were mounted with Moviol. Cells were observed using Nikon Eclipse 50I microscopy and images were captured using Nis-Elements D 4.00 software (Nikon Instruments Europe BV-Netherlands).
Data were displayed and analyzed using Adobe® Photoshop®CS4.
The MITO CytoPainter mitochondrial indicator is a hydrophobic compound that easily permeates intact live cells and becomes trapped in mitochondria after it enters the cells. This staining was performed on C2C12 live cells during the proliferative phase and all the differentiation phases.
Cell ROX® Oxidative Stress Reagents are fluorogenic probes designed to reliably measure ROS in live cells. The cell-permeable reagents are non-fluorescent or very faintly fluorescent while in a reduced state, and during oxidation exhibit a strong fluorogenic signal. Cell ROX® Orange Reagents are localized in the cytoplasm. This staining was performed on C2C12 live cells during the proliferative phase.
Slides were mounted with Moviol. Cells were observed using Nikon Eclipse 50I microscopy and images were captured using Nis-Elements D 4.00 software (Nikon Instruments Europe BV-Netherlands).
Data were displayed and analyzed using Adobe® Photoshop®CS4.
9
Mitochondrial Dynamics and Oxidative Stress in hObs
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After having starved hObs for 24h, cells were treated for 3, 6, and 24 h with L-C (5 mM). hObs, fixed and permeabilized, were incubated in PBS with 1% BSA to avoid nonspecific binding of the antibody. Cells were then incubated with specific antibodies FITC or rhodamine conjugated, and nuclei were revealed with DAPI staining. The MITO CytoPainter mitochondrial indicator is a hydrophobic compound that easily permeates intact live cells and becomes trapped in mitochondria. Cell ROX® Oxidative Stress Reagents are fluorogenic probes designed to measure reactive oxygen species (ROS) in living cells that exhibit a strong fluorogenic signal during oxidation. Cells were analyzed using Nikon Eclipse 50I microscopy with Nis-Elements D 4.00 software (Nikon Instruments Europe BV, Netherlands). Quantification of immunofluorescence signal was performed by using Image J program (http://imagej.nih.gov/ij/).
10
Immunostaining of Tissue Sections and Cells
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For tissues analysis, 7 μm frozen cryosections was fixed in Formaldehyde 4% overnight at 4C. After that, slides were washed in PBS and incubated for 1 hour at room temperature with 10% horse serum in PBS with 0.05% Triton X-100 to block nonspecific binding sites, while C2C12 cells, fixed and permeabilized as described [22 (link)], were blocked with PBS containing 1% bovine serum albumin. Slides or cells were then immunostained with specific antibodies rhodamine-conjugated and nuclei revealed with DAPI staining. Slides were mounted with Moviol. Cells were observed using Nikon Eclipse 50I microscopy and images were captured using Nis-Elements D 4.00 software (Nikon Instruments Europe BV, Netherlands). Data were displayed and analyzed using Adobe Photoshop CS4. Live C2C12 cells were examined and images were acquired by phase contrast microscopy using the same microscope and digital system described above.
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