The nucleotide sequences of three genes, that is, the largest and second largest subunits of RNA polymerase II (RPB1, RPB2) and translation elongation factor 1α (EF1-α), were either obtained from our sequenced PCR products of the O. formosana isolates, retrieved from the NCBI database following the criteria described by Sung et al. [21 (link)], or acquired by whole genome ortholog BlastNs of C. militaris CM01. A total of 51 species/isolates were included in this study (Supporting Table 3). The nucleotide sequences of these three genes were concatenated, aligned, and tested in models using MEGA 6.0 [28 (link)]. The best model according to the AIC and BIC values was the GTR+I+G model. The aligned sequences were analyzed using CIPRES portal-based Raxml blackbox (RAxML 7.2.7) [29 (link)] for maximum likelihood (ML) tree searching with the default settings, an estimation of the gamma distribution, invariable sites, and Bayesian inference of the phylogeny with MrBayes 3.2.3 with the parameters obtained from the model test. The resulting phylogenetic trees were saved and plotted using Adobe Illustrator CS3.
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Lab products found in correlation
Lsm 780, by Zeiss (4 mentions)
Microfire digital camera, by MBF Biosciences (2 mentions)
Lasersharp 2000, by Bio-Rad (2 mentions)
Velocity software, by PerkinElmer (2 mentions)
Radiance 2100 rainbow, by Nikon (2 mentions)
Zen software, by Zeiss (2 mentions)
Nis elements software, by Nikon (2 mentions)
Axiocam, by Zeiss (2 mentions)
Lsm 510 meta, by Zeiss (2 mentions)
Axio imager, by Zeiss (2 mentions)
Lsm 700, by Zeiss (2 mentions)
Prism 6, by GraphPad (2 mentions)
Alexa 488, by Thermo Fisher Scientific (2 mentions)
Microsoft office powerpoint 2007, by Adobe (2 mentions)
Anti β tubulin 3, by Abcam (1 mentions)
Streptavidin alexa fluor 647, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 594, by Thermo Fisher Scientific (1 mentions)
3 3 diaminobenzidine (dab), by Vector Laboratories (1 mentions)
Vectastain elite abc kit, by Vector Laboratories (1 mentions)
77 protocols using illustrator cs3
1
Phylogenetic Analysis of Fungal Genes
2
Immunochemistry of Bacterial Infection
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Animal tissue was prepared and cryostat sections were cut as previously described (31 (link)), and immunochemistry was performed as previously described (32 (link)). The primary antibodies used were rabbit anti-B. pseudomallei (1:2,000) (33 (link)), polyclonal goat anti-olfactory marker protein (OMP; 1:1,000; Wako), and polyclonal rabbit anti-β-tubulin III (1:500; Abcam) followed by the secondary antibody anti-rabbit Alexa Fluor 488 or anti-goat Alexa Fluor 594 (1:400; Invitrogen). The biotinylated lectin UEA1 (1:100; Dako) was incubated similarly to the antibodies, followed by streptavidin-Alexa Fluor 647 (1:400; Invitrogen). Cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). Images were captured using an Olympus FV1000 microscope, and image panels were created using Adobe Illustrator CS3.
3
Immunostaining of Enteric Neurons and Macrophages
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As previously reported, DA neurons and macrophages of the myenteric plexus were identified on free-floating sections following immunohistochemistry (Côté et al., 2011 (link)). Briefly, myenteric ganglia neurons were stained with Cuprolinic blue for 60 min at 37°C (Holst and Powley, 1995 (link)). DA neurons and macrophages were identified with a polyclonal tyrosine hydroxylase antibody (TH; 1:1000; Cedarlane, ON, Canada) and the pan-macrophage/microglia marker ionized calcium-binding adaptor molecule 1 (Iba-1; 1:1000; Cedarlane), respectively. Biotinylated goat anti-rabbit IgG (1:1000; Cedarlane) was used as a secondary antibody. The signal was revealed with the ABC Vectastain Elite kit (Vector Laboratories, Inc., ON, Canada) and 3,3′-diaminobenzidine (DAB, Vector Laboratories, Inc.) as chromagen. All pictures were acquired with a Nikon C80i microscope equipped with a MicroFire digital camera (MBF Bioscience, Williston, VT). Figures were assembled using Adobe Illustrator CS3.
4
Immunofluorescence Staining of Bladder Tissue
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Excised bladders were fixed in 4% w/v paraformaldehyde dissolved in 100 mM sodium cacodylate (pH 7.4) buffer for 2 h at room temperature. Fixed tissue was cut into small pieces with a razor blade, cryoprotected, frozen, and sectioned. Cells grown on collagen-coated glass coverslips were fixed for 20 min with 4% (w/v) paraformaldehyde. Tissue or cells were incubated with primary antibodies (1:100) for 2 h at room temperature. After washing away unbound primary antibody, sections were incubated with an Alexa 488-conjugated secondary antibody (diluted 1:100), and actin was stained with rhodamine phalloidin (Cytoskeleton, PHDR1). Tissue or cells were mounted with SlowFade Diamond Antifade Reagent containing DPAI (Thermo Fisher Scientific) to stain nuclei. Images acquired by Zeiss LSM-510 confocal microscope were saved as TIFF files, and imported into Adobe Illustrator CS3. Additional antibodies included rat anti-mouse β1 integrin antibody (BD Bioscience, #550531), anti-c-fos (Cell Signaling, #2250), anti-c-jun (Cell Signaling, #9165), and anti-Ki67 (Abcam, #ab15580).
5
Multimodal Imaging and Co-localization Analysis
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Immunofluorescence staining and microscopic observations were performed as previously described (Jacob et al., 2009 (link)). Different fluorescence signals were imaged by using a laser scanning confocal microscope (Zeiss LSM 510 META). Images were processed with Illustrator CS3 and Photoshop CS3 (Adobe). Co-localisation analysis was performed using Imaris (Bitplane).
6
Quantifying Diabetes and Insulitis in Mice
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Diabetes and insulitis were assessed as previously described (Paterson et al., 2011 (link)). Briefly, mice were monitored weekly for high urine glucose by Diastix (Diastix; Bayer). Mice positive by Diastix were confirmed by blood glucose measurement (Ascensia Contour; Bayer). Diabetes was confirmed by blood glucose level of ≥250 mg/dl. For histology, pancreata were fixed in 10% buffered formalin overnight, embedded in paraffin, and stained with hematoxylin and eosin. Islets were scored in blinded fashion as follows: 0, no infiltration; 1, perivascular/periductular infiltrates with lymphocytes touching islet perimeters, but not penetrating; 2, lymphocytic penetration of up to 25% of islet mass; 3, lymphocytic penetration of up to 75% of islet mass; and 4, <20% of islet mass remaining. Photomicrographs were taken with a mounted digital camera (Olympus DP71) driven by Olympus DP Controller software. Images were prepared using Adobe Photoshop and Illustrator CS3 (Adobe Systems).
7
Scanning Electron Microscopy of Cockroach Antennae
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After anesthetising nymphal and adult cockroaches on ice, whole antennae were isolated using a razor blade. The isolated antennae were immersed in 50% acetone and ultrasonically cleaned for 1 min. Antennae were dehydrated in an ascending acetone series (50% to 100%) and dried at 60 °C for more than 3 hours. Thereafter, each antenna was attached to an aluminium stub using water-soluble glue. After drying again, antennae were coated with platinum-palladium using an ion sputter (E-1045; Hitachi, Tokyo, Japan). Observations were performed using a field emission SEM (S-4800; Hitachi, Tokyo, Japan). After counting the number of flagellomeres, the external structures of the sensilla on a given flagellomere were thoroughly examined, and their digital images were obtained. The obtained images were processed using Adobe Photoshop CS3 and Illustrator CS3 (Adobe Systems, San Jose, CA, USA). Regarding the sensillar nomenclature, we referred to previous studies of adult P. americana15 (link),17 (link),18 ,28 (link).
8
Image Processing and Visualization Protocol
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Global contrast and brightness values of some of the images were adjusted using Adobe Photoshop CS3. Figures were compiled in Adobe Illustrator CS3. If not stated otherwise, anterior is (1) to the top in all ventral or dorsal aspects and in horizontal sections and (2) to the left in lateral aspects and sagittal sections. In anterior or posterior aspects and transverse sections, dorsal is to the top.
Short movies (avi-format) were generated in Imaris, using the ‘Animation’ mode. They were down-sized to compressed avi-files with the freeware FormatFactory (Version 2.96).
Short movies (avi-format) were generated in Imaris, using the ‘Animation’ mode. They were down-sized to compressed avi-files with the freeware FormatFactory (Version 2.96).
9
Expansin Gene Structure and Motif Analysis
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Analysis of gene structure was performed to identify exons, introns, and UTRs. The corresponding GFF data of identified expansin gene IDs were extracted from the GFF file named Ghirsutum_gene_model in the new cotton genome data [19 (link)] (http://cotton.hzau.edu.cn/EN/download.php ), and then the expansin GFF data were analysed using the online tool GSDS (version 2.0, http://gsds.cbi.pku.edu.cn/ ) [38 (link)]; the results were saved in SVG image format. Motifs of expansin protein sequences were analysed using the online tool MEME (http://meme-suite.org/index.html ) [39 (link)]. According to the required file format, we imported the expansin sequences into the online tool. The maximum number of motifs was set to 10, the repeat number was set to 0 or 1, and the remainder of the parameters were set to system defaults. The output draft images of gene structure and motif were further modified with Adobe Illustrator CS3 software (version 13.0.0).
10
Multimodal Fluorescence Microscopy Imaging
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Images were captured on a fluorescence microscope (Nikon E90i or Nikon E1000) equipped with a Nikon Intensilight C-HGFI source or an X-Cite 120 illuminator (EXFO), respectively, using high numerical aperture optics and a cooled CCD camera (ANDOR or Q-Imaging, respectively) running NIS Elements software (Nikon) or Velocity software (Improvision, Lexington, MA).
Five-channel confocal imaging was performed at the Harvard Center for Biological Imaging on a Zeiss 710 inverted confocal microscope equipped for spectral unmixing and for acquisition of confocal montages of entire tissue sections, running ZEN software (Zeiss). Other confocal images were obtained using a BioRad Radiance 2100 Rainbow laser-scanning confocal system based on a Nikon E800 microscope with LaserSharp 2000 imaging software (Bio-Rad Laboratories). Some images were subsequently processed for 3D reconstruction in Imaris (Bitplane). For an optimal visual reproduction of the data, images were adjusted for contrast, brightness, color balance, and size in Photoshop CS3 (Adobe, San Jose, CA), and assembled in Power Point for Mac 2011 (Microsoft Corporation, Redmond, WA), and in Illustrator CS3 (Adobe, San Jose, CA).
Five-channel confocal imaging was performed at the Harvard Center for Biological Imaging on a Zeiss 710 inverted confocal microscope equipped for spectral unmixing and for acquisition of confocal montages of entire tissue sections, running ZEN software (Zeiss). Other confocal images were obtained using a BioRad Radiance 2100 Rainbow laser-scanning confocal system based on a Nikon E800 microscope with LaserSharp 2000 imaging software (Bio-Rad Laboratories). Some images were subsequently processed for 3D reconstruction in Imaris (Bitplane). For an optimal visual reproduction of the data, images were adjusted for contrast, brightness, color balance, and size in Photoshop CS3 (Adobe, San Jose, CA), and assembled in Power Point for Mac 2011 (Microsoft Corporation, Redmond, WA), and in Illustrator CS3 (Adobe, San Jose, CA).
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