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667 protocols using Illustrator CS6

Light micrographs were adjusted in Adobe Photoshop CS6 for contrast and assembled in Adobe Illustrator CS6. All figures and drawings were prepared in Adobe Illustrator CS6.
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2

Detailed Visualization and Statistical Analysis Methods

Illustrations were prepared using PowerPoint (Microsoft) and Illustrator CS6 (Adobe). Bar graphs, dose-response graphs and circle graphs were prepared using Prism 7 (GraphPad Software) and statistical analysis was performed using the same software. For comparisons between two datasets, significance was determined by unpaired t test; for comparisons between more than two datasets, significance was determined by one-way ANOVA. Significance is indicated as **** (p<0.0001), ** (p<0.01), * (p<0.05) or ns (not significant). Pictures of gels and immunoblots were only adjusted for contrast and brightness using Photoshop CS6 (Adobe) and were arranged in Illustrator CS6.
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Mounted specimens were scanned in Leica SP5 confocal laser scanning microscope. Z- stacks of scans were projected into 2D images and 3D reconstructions in IMARIS 9.1.2, which was also used to conduct all the measurements. Schematic drawings based on Z- stacks of scans were made in Adobe Illustrator CS6. Additionally, some living animals anesthetized with bupivacaine solution were photographed with Zeiss Axiocam HRc connected to a Zeiss Axioscope Ax10 using bright-field Nomarski optics. CLSM and light microscopy images were adjusted in Adobe Photoshop CC 2015 and assembled in Adobe Illustrator CS6.
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Illustrations were prepared using PowerPoint (Microsoft) and Illustrator CS6 (Adobe). Circle plots depicting the hits from each screen, GT insertion histograms, dose response graphs, tables and supplementary files were prepared using Excel (Microsoft). Bar and circle graphs were prepared using Prism 6 (GraphPad Software) and statistical analysis was performed using the same software (details of statistical tests used are given in the figure legends). Heat maps were generated using Partek Genomics Suite 6.6 software (Partek Inc.) and finished in Illustrator CS6. FACS histograms and dot plots were generated using FlowJo (FlowJo, LLC) and finished in Illustrator CS6. Pictures of immunoblots and model organisms were only adjusted for contrast and brightness when necessary for clarity using Photoshop CS6 (Adobe), and were arranged in Illustrator CS6.
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5

Comparative Genomics and Developmental Expression of Nodal and Pitx

Full-length sequences of nodal in N. anomala, and Pitx in O. fusiformis, L. ruber and P. caudatus were identified from RNAseq data of mixed embryonic stages. Protein alignments were constructed with MAFFT v. 7 [51 (link)] and poorly aligned regions were removed with Gblocks v. 0.91b [52 (link)]. RAxML v. 8 [53 (link)] was used to infer gene orthologies (electronic supplementary material, figure S1). Resulting trees were formatted with FigTree and Illustrator CS6 (Adobe). Fixed embryos of N. anomala, O. fusiformis, L. ruber and P. caudatus were used to perform colorimetric whole mount in situ hybridization following previously described protocols [46 (link),49 (link)]. After developing the signal, samples were stored in 70% glycerol and imaged with an Axiocam HRc connected to an Axioscope Ax10 (Zeiss), using bright field Nomarski optics. Images were analysed with Photoshop CS6 (Adobe), and figure plates made with Illustrator CS6 (Adobe). Contrast and brightness were adjusted always to the whole image and not to specific parts of it.
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Samples for confocal laser scanning microscopy (antibody staining and in situ hybridization) were mounted in Murray’s clear and scanned in either Leica SP5 or Olympus FV3000 CLSM. Z-stacks of confocal scans were projected into 2D images in IMARIS 9.1.2. TEM microphotographs were obtained with Gatan ES500W camera mounted on transmission electron microscope Jeol JEM-1011. Both CLSM images and TEM micrographs were assembled in Adobe Illustrator CS6 into final figures. All the schematic drawings were done with Adobe Illustrator CS6.
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Sections for LM were examined with a Leica DMR microscope equipped with a digital camera (Leica DFC-450). Photopanels were constructed with Adobe Photoshop and Illustrator CS6. The 3D-plots of the spinal cords of Figures 3D1–3 were made using a motorized Olympus BH microscope equipped with a Lucivid miniature monitor and NeurolucidaTM software (Microbrightfield). Cells were determined to be motoneurons if they were located within the lateral motoneurons column, had a diameter of at least 25 μm. Cells were only plotted if they contained a nucleus. RABV deposits reflecting lytic motoneurons were plotted if the size of the deposit was at least 40 μm. The Neurolucida set-up was also used to count and measure contours of labeled neurons. Diagrams were constructed with ExcelTM (Microsoft Office 2010).
Fluorescent labeling was assessed using either a Leica DMRBE microscope and appropriate filters and photographed with a Hamamatsu camera (C4880) or with a Zeiss LSM700 confocal microscope and Zeiss 2009 software (ZenTM). Photopanels were constructed in Adobe Photoshop and Illustrator CS6.
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Z-stacks of confocal scans were projected into 2D images and 3D reconstructions in IMARIS 9.1.2. Both light micrographs and CLSM images were adjusted in Adobe Photoshop CS6 and assembled in Adobe Illustrator CS6. All the schematic drawings were done with Adobe Illustrator CS6.
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For > 6 months after the procedure, the recipient site was observed for graft take, presence of secondary contraction, and color and texture match. Secondary contraction in particular was checked by tracing and scanning the graft skin immediately after the procedure and at 6 months (or more) after the operation and outlining the data on Adobe Illustrator CS6. A plug-in computer software “Hakariya” (Comnet Co., Ltd., Kobe, Japan), which can be used for automatically calculating square measure, circumference and length, and settings of scales on Adobe Illustrator CS6 was downloaded. The square measures of the graft skin were calculated according to the set scale, thus contraction rate was confirmed (Fig. 1). Contraction percentages were defined as follows: <5% : “no contraction,” 5%–30% : “mild contraction,” and >30% : “contraction.”
Regarding color and texture match of the recipient site, results were defined as follows: good: mild pigmentation/both patient and physician are satisfied; fair: mild pigmentation/either patient or physician is dissatisfied; and poor: severe pigmentation.
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All values were expressed as mean ± SD. Data were analyzed by one-way variance (ANOVA) followed by Tukey’s multiple comparison with SPSS 25 software (IBM SPSS Statistics 25.0, Armonk, NY, USA). The p < 0.05, p < 0.01 and p < 0.001 were set at the threshold for statistical significance, high statistical significance, and very high statistical significance, respectively. Graphs were constructed using GraphPad Prism 8 (GraphPad Prism 8.0, San Diego, CA, USA) and Adobe illustrator CS6 software (Adobe illustrator CS6, San Jose, CA, USA).
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