HEK293 or Saos-2 cells were transfected with control vector, Myc-EBNA3C or Flag-METTL14 and allowed to grow in DMEM supplemented with 1mg/ml G418 (Sigma-Aldrich, St. Louis, MO, USA). After two weeks of selection, 105 cells were seeded in 6-well plates and allowed to grow for 5 days. Cells were stained with 0.005% Crystal Violet overnight and scanned by PhosphorImager (Molecular Dynamics, Piscataway, NJ) and the area of the colonies measured by using Image J software (Adobe Inc., San Jose, CA). Three independent experiments were performed. The soft agar assays were performed using LcLs and Burkitt lymphoma cells. Briefly, 1 ml of 0.5% agar in RPMI media was poured into 6-well plates and set aside to solidify. 0.5 ml 0.3% agar/medium containing 2×105 cells were added to the plates as the middle layer. Then cells were covered with a top layer of another 1ml 0.5% agar/medium. After two weeks, colonies were stained with 0.005% crystal violet for 1 hour and scanned using a Licor Odyssey system (LiCor Inc., Lincoln, NE). The number of colonies was counted using ImageJ software (Adobe Inc., San Jose, CA).
Image j software
Manufactured by Adobe
Sourced in United States
ImageJ is a free and open-source image processing software. It provides a platform for viewing, analyzing, and processing digital images and micrographs. The software's core function is to enable users to perform a wide range of image-related tasks, including image enhancement, measurement, and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 710, by Zeiss (6 mentions)
Axiocam mrc5 camera, by Zeiss (5 mentions)
Axio imager m1 compound microscope, by Zeiss (3 mentions)
Lsm 710 confocal microscope, by Zeiss (3 mentions)
Axio imager m1 compound, by Zeiss (2 mentions)
Crystal violet solution, by Merck Group (2 mentions)
Phosphorimager, by GE Healthcare (2 mentions)
Lasso tool, by Adobe (2 mentions)
Zen imaging software, by Adobe (2 mentions)
Lsm 700, by Zeiss (2 mentions)
Lsm 510, by Zeiss (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Dp71 digital camera, by Olympus (1 mentions)
Odyssey system, by LI COR (1 mentions)
Illustrator 2020, by Adobe (1 mentions)
Prism 9, by GraphPad (1 mentions)
Dulbecco s pbs, by Thermo Fisher Scientific (1 mentions)
Cell recovery solution, by Corning (1 mentions)
D2522, by Merck Group (1 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (1 mentions)
31 protocols using image j software
1
Assessing Oncogenic Potential of Proteins
2
Statistical Analysis of Experimental Data
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The data were presented as mean ± SD for at least three different determinations. Statistical analysis was performed with the SPSS version 21. The data were analyzed by GraphPad Prism software, Image J software, and Adobe Photoshop CS6 (San Jose, CA, USA). Differences between variants were analyzed by the Student's t test or one-way ANOVA. ∗P < 0.05 ∗∗P < 0.01 were considered statistically significant.
3
Microscopic Imaging of Spinal Cord Embryos
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Embryos were mounted in 70% glycerol:30% distilled water and Differential Interference Contrast (DIC) pictures were taken using an AxioCam MRc5 camera mounted on a Zeiss Axio Imager M1 compound microscope. Fluorescent images were taken on a Zeiss LSM 710 confocal microscope.
Images were processed using Adobe Photoshop software (Adobe, Inc) and Image J software (Abramoff et al., 2004) . In some cases, different focal planes were merged to show labeled cells at different mediolateral positions in the spinal cord. All images were processed for brightness-contrast and color balance using Adobe Photoshop software (Adobe, Inc.). Images of control and mutant embryos from the same experiment were processed identically. Figures were assembled using Adobe Photoshop and Adobe Illustrator (Adobe, Inc.).
Images were processed using Adobe Photoshop software (Adobe, Inc) and Image J software (Abramoff et al., 2004) . In some cases, different focal planes were merged to show labeled cells at different mediolateral positions in the spinal cord. All images were processed for brightness-contrast and color balance using Adobe Photoshop software (Adobe, Inc.). Images of control and mutant embryos from the same experiment were processed identically. Figures were assembled using Adobe Photoshop and Adobe Illustrator (Adobe, Inc.).
4
Semiquantitative Immunohistochemical Analysis of Kidney Structures
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We used three samples per group and 10 sections per each sample. The analysis was performed with the fluorescence microscope (Olympus BX61, Tokyo, Japan) equipped with a DP71 digital camera (Olympus). Images were analyzed with ImageJ Software and Adobe Photoshop (Adobe, San Jose, CA, USA). The staining intensity of kidney structures was semiquantitatively organized into four groups: the absence of any reactivity (−), mild reactivity (+), moderate reactivity (++), and strong reactivity (+++) (Table 2 ). The percentage of DAB1 and Reelin immunoreactive cells was calculated. For each investigated period, we captured at least twenty images per different kidney structure: proximal convoluted tubules (PCT), distal convoluted tubules (DCT), and glomeruli at 40 × objective magnification. We averaged the number of positive cells in each kidney structure from 10 sections. Any level of cytoplasmic or membrane staining with the used markers was regarded as positive. Two investigators analyzed the images independently.
5
KSHV-Driven Colony Formation Assay
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For colony formation and soft agar assays, KSHV positive BC3 cells were transduced with lentivirus coding either ShControl or ShvCyclin. The transduced cells were selected with puromycin for 3 weeks. 100% stably transduced (GFP positive) cells were used for colony formation and soft agar assay. Ten million HEK293T-KSHV cells were transfected with pGIPZ-shCon, shHIF1α or shvCyclin plasmids by electroporation and seeded in 10 cm dishes. Transfected cells were grown in DMEM containing puromycin at 0.5 µg/ml concentration. After selecting the cells for up to 2 weeks, selected cells were fixed with 4% formaldehyde and stained with 0.1% Crystal Violet solution (Sigma-Aldrich Corp., St. Louis, MO). The area of the colonies was calculated by using Image J software (Adobe Inc, San Jose, CA). The data shown here represents the average of three independent experiments.
6
Quantifying Retinal Morphology and Vascularization
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Morphometric analyses of the retinas were performed using Image J software and Adobe Photoshop software. To determine the amount of regression and neovascularization, the number of pixels in the avascular regression area and neovascular area was measured using the Lasso tool of Adobe Photoshop software as described 54 (link), and divided by the number of pixels in the total retinal area and presented as a percentage. RBC leakage was measured as TER119-stained area outside the vessels divided by total measured area of retina as previously described 70 (link). Macrophage infiltration was measured as the total number of F4/80+ macrophage in the whole retina.
7
Imaging Embryonic Spinal Cord Development
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Embryos were mounted in 70% glycerol:30% distilled water and differential interference contrast (DIC) pictures were taken using an AxioCam MRc5 camera mounted on a Zeiss Axio Imager M1 compound microscope. Fluorescence images were taken on a Zeiss LSM 710 confocal microscope. Images were processed using Adobe Photoshop software (Adobe, Inc) and Image J software (Abramoff et al. 2004). In some cases, different focal planes were merged to show labeled cells at different medio-lateral positions in the spinal cord. All images were processed for brightness, contrast and color balance using Adobe Photoshop software (Adobe, Inc.). Images of control and mutant embryos from the same experiment were processed identically. Figures were assembled using Adobe Photoshop and Adobe Illustrator (Adobe, Inc.).
8
Validating Dual-Band Fluorescence for Labeling
9
Robust Statistical Analysis of Experimental Data
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All data were analyzed using GraphPad Prism 9.0 (GraphPad, San Diego, CA, USA) and expressed as mean ± SD. Data were evaluated by unpaired Student’s t-test between two independent groups. Survival curves were assessed by log-rank (Mantel-Cox) tests. P < 0.05 was considered significant. Adobe Illustrator 2020, Adobe Photoshop 2020, and Image J software were used for the stitching of pictures.
10
Visualization of Receptor Internalization in HEK-293 Cells
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To study receptor internalization, HEK-293 cells stably transfected with cMyc-δOR/HA-SSTR4 were treated with SST-14 (1 µM), SB-205607 (10 nM) alone or in combination for 15 min at 37°C. Treatment was terminated by washing with ice cold Dulbecco’s PBS (GIBCO, Invitrogen, Burlington, ON, Canada). Cells were then fixed with 4% paraformaldehyde for 20 min on ice. To visualize intracellular expression of the receptor, cells were permeabilized with 0.2% Triton X-100 for 10 min and processed for immunocytochemistry as earlier reported [42] (link), [45] (link). Merged images showing colocalization were generated by using NIH, ImageJ software and the photograph composites were made by using Adobe Photoshop (San Jose, CA). Quantification of immunofluorescence intensity was performed by using NIH, ImageJ software.
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