Publication quality images of injection sites and labeled corticospinal nerve fibers and terminals were captured using a Spotflex 64 Mp camera, (Diagnostic Instruments Inc., Sterling Heights, MI, USA, version 4.6), mounted on an Olympus BX51 microscope. Photographic montages of the injection sites and labeled fibers were created using Adobe PhotoShop 7.0 (Adobe Systems Inc., San Jose, CA, USA). Only brightness and contrast were adjusted to maximize discrimination and normalize images for comparative purposes. Cortical surface reconstructions and ICMS maps were developed as previously described using metrically calibrated digital images of the cortical surface (Morecraft and Van Hoesen 1992 (link), Morecraft et al., 2013 (link)). Density plots of terminal boutons were made using our Neurolucida microscope workstation to illustrate the topographical distribution of the iCSP from M1 in the controls and lesion cases (Fig. 5 ). Publication quality line illustrations were created using Adobe Illustrator 10.0 (Adobe Systems Inc., San Jose, CA, USA).
Illustrator 10
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Sourced in United States
Adobe Illustrator 10 is a vector graphics software application used for creating, editing, and manipulating digital illustrations, logos, and artwork. It provides a range of tools and features for working with vector-based graphics, allowing users to create, manipulate, and refine their designs with precision.
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Lab products found in correlation
Bx51 microscope, by Olympus (3 mentions)
Originpro 8, by OriginLab (2 mentions)
Clampfit 10, by Molecular Devices (2 mentions)
Creative suite 5, by Adobe (2 mentions)
Sigmaplot, by Grafiti LLC (2 mentions)
Qiaquick gel extraction kit, by Qiagen (1 mentions)
Jmp pro 10, by SAS Institute (1 mentions)
Mouse anti neun, by Merck Group (1 mentions)
Mouse anti gfap, by Merck Group (1 mentions)
Goat anti rabbit rhodamine conjugated secondary antibody, by Merck Group (1 mentions)
Hybond c, by Cytiva (1 mentions)
Nupage, by Thermo Fisher Scientific (1 mentions)
Ecl system, by Cytiva (1 mentions)
Microcal origin 6, by Malvern Panalytical (1 mentions)
Prism version, by GraphPad (1 mentions)
Western blotting luminol reagent, by Santa Cruz Biotechnology (1 mentions)
Criterion tris hcl precast gel, by Bio-Rad (1 mentions)
Tris buffered saline (tbs), by Bio-Rad (1 mentions)
Imagequant las 4000, by GE Healthcare (1 mentions)
E600 microscope, by Nikon (1 mentions)
17 protocols using illustrator 10
1
Microscopic Imaging and Reconstruction of Cortical Networks
2
Microscopy Imaging and Cortical Reconstruction
3
Electrophysiological Data Analysis Methods
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SPSS 17.0 software was used for the data analysis (SPSS, Chicago, IL, USA). Clampfit 10.6 (Molecular Devices, USA), OriginPro 8.0 (Origin Lab, USA), and Adobe Illustrator 10.0 (Adobe, USA) were used to analyze the patch-clamp recorded data. All the data are expressed as the means ± standard errors of the means. A one-way analysis of variance followed by a multiple-comparison test was used to evaluate the multiple test treatments. A P value <0.05 was considered statistically significant. In the figures, the designations for the P values are as follows: *, P<0.05, **, P<0.01, and ***, P<0.001 respectively.
4
Cortical Mapping Using Photographic Techniques
5
Phylogenetic Analysis of β-giardin Sequences
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Bands were excised from agarose gels and purified using the QIAquick Gel Extraction Kit (Qiagen, GmbH. Germany) according to the manufacturer’s instructions. DNA sequencing was conducted in both directions using the PCR primers (Iontek Inc., Turkey). The sequences (23 of them) were contig assembled in vector NTI (Life Tech, USA), edited in BIOEDIT, and used in BLAST search for identification of assemblages [13 (link)]. The results revealed that all sequences belonged to β-giardin.
The sequences were then aligned and examined. Examination of the alignment revealed the presence of partial sequences and the sequences that generated dubious quality. These sequences or sequence parts were systematically eliminated. The edited sequences were then aligned with Clustal W using default parameters and were subjected to phylogenetic analysis using the freely available PHYLIP package [14 ]. In brief, the sequence data were bootstrapped 1000 times by randomly choosing columns from the original alignment by using the SEQBOOT program. The input order of the sequences was randomized with a jumble number of 10. Then NJ (NEIGHBORJOINING) trees were built by using the generated bootstrapped data. The majority rule consensus trees were created by using CONSENSUS and the tree was drawn with DRAWTREE and edited in Adobe Illustrator 10.
The sequences were then aligned and examined. Examination of the alignment revealed the presence of partial sequences and the sequences that generated dubious quality. These sequences or sequence parts were systematically eliminated. The edited sequences were then aligned with Clustal W using default parameters and were subjected to phylogenetic analysis using the freely available PHYLIP package [14 ]. In brief, the sequence data were bootstrapped 1000 times by randomly choosing columns from the original alignment by using the SEQBOOT program. The input order of the sequences was randomized with a jumble number of 10. Then NJ (NEIGHBORJOINING) trees were built by using the generated bootstrapped data. The majority rule consensus trees were created by using CONSENSUS and the tree was drawn with DRAWTREE and edited in Adobe Illustrator 10.
6
Comparative Statistical Analyses of Experiments
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All samples were assayed and quantified in randomized order and group assignments were made after final data curation. One-way ANOVAs with Tukey post hoc test, or unpaired t-test, was used for parametric continuous variables to compare group means. Kruskal-Wallis one-way analysis with Dunn test for post hoc comparison, or Mann-Whitney U test, were used to compare median values of categorical and nonparametric variables. Correlation analyses were performed using Spearman Rank-Order tests to accommodate nonparametric variables. All significance levels are reported as two-tails, corrected for multiple testing, with p <0.05 interpreted as significant. Statistical analysis was performed using JMP Pro 10 (SAS Institute Inc), graphs built with GraphPad Prism 5.0 and arranged in Adobe Illustrator 10.
7
Subsidence Analysis of Barapukuria Coal Mine
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From the bird’s eye view of the Barapukuria coal mine area, the subsided area can be divided into two regions (Fig. 6 a), i.e., the northern and southern parts considering the subsidence epicenters22 . The subsidence in the north is just above the Northern Upper Panels and is named NUP, and it is above the Southern Lower Panels in the south, named SLP. The observed subsidence is shown as a contour map in Fig. 6 b23 . The subsidence in the north can be further subdivided into North-Western and North-Eastern zones. The maximum subsidence in the North-Western and the North-Eastern zones is 5.8 m and 4.6 m, respectively (Fig. 6 b), whereas; it is 4.2 m in the southern part. The observed subsidence of the contour map was converted to grid values having a specific range in the modeled grid area to compare the observed and the predicted subsidence.![]()
The subsidence in the Barapukuria coal mine area. (
8
Immunofluorescence Staining of Mixed Cortical Cells
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Living mixed cortical cells were stained for 1 h at 4 °C using rabbit polyclonal anti-PKR1 or anti-PKR2 antibodies 1:400 in PBS (Alomone Labs, Jerusalem, Israel), washed in PBS and incubated with a goat anti-rabbit rhodamine-conjugated secondary antibody (Sigma) for 30 min at room temperature. This procedure allowed us to specifically target surface protein expression. Cells were then fixed in 4% (w/v in PBS) paraformaldehyde for 10 min at room temperature on immunofluorescence-labeled coverslips. For PROK2 immunofluorescence, CNs were first fixed in 4% (w/v in PBS) paraformaldehyde following the above procedure, incubated with rabbit polyclonal anti-PROK2 antibody (Abcam, Cambridge, UK 1:400), washed in PBS and incubated with a goat anti-rabbit rhodamine-conjugated secondary antibody (Sigma) for 30 min at room temperature.
CNs cells were then incubated with mouse anti-NeuN (Sigma, 1:200 dilution) or mouse anti-GFAP (Sigma, 1:400 dilution) overnight at 4 °C and with a goat anti-mouse alexa-488 conjugated secondary antibody (Sigma, 1:400) for 30 min at room temperature. For nuclei visualization, coverslips were incubated with Hoecsht 33258 (0,25 μg/ml) for 5 min at room temperature. Cells were visualized by a confocal laser scanning microscope (Leica SP5, Leica Microsystems, Wetzlar, Germany). Final figures were assembled by using Adobe Photoshop 7 and Adobe Illustrator 10.
CNs cells were then incubated with mouse anti-NeuN (Sigma, 1:200 dilution) or mouse anti-GFAP (Sigma, 1:400 dilution) overnight at 4 °C and with a goat anti-mouse alexa-488 conjugated secondary antibody (Sigma, 1:400) for 30 min at room temperature. For nuclei visualization, coverslips were incubated with Hoecsht 33258 (0,25 μg/ml) for 5 min at room temperature. Cells were visualized by a confocal laser scanning microscope (Leica SP5, Leica Microsystems, Wetzlar, Germany). Final figures were assembled by using Adobe Photoshop 7 and Adobe Illustrator 10.
9
Experimental Protocol for Differential Analysis
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Unless otherwise indicated, all p-values were obtained with wilcox.test and alternative = "greater". We used “Hochberg” method for adjusted P-value. multiple correction method (Table S4 ). In all boxplots, center lines indicate median values and box heights indicate the inter-quartile range of data. The ggplot2 library from R software version 3.6.1 (2019-07-05) was used for plotting all plots and figures. Adobe Illustrator 1.0 was used for formatting all figures.
10
Western Blot Quantification Protocol
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Equal amounts of protein were subjected to SDS-PAGE 7.5-15% linear gradient or Bis- Tris gel 4-12% (NuPage, Invitrogen). After electroblotting onto a nitrocellulose membrane (Hybond-C Amersham Biosciences, Piscataway, NJ) the filters were blocked in TBS containing 10% non-fat dried milk for 1h at room temperature or overnight at 4°C. Proteins were visualized using appropriate primary antibodies. All primary antibodies were diluted in TBS and incubated with the nitrocellulose blot overnight at 4°C. Incubation with secondary peroxidase coupled anti-mouse, anti-rabbit or anti-goat antibodies was performed by using the ECL system (Amersham, Arlington Heights, IL, U.S.A.) In a few experiments, multiple normalizations of the same filter on different loading controls, such as β-actin and GAPDH (glyceraldehyde 3-phosphate dehydrogenase), were carried. Final figures were assembled by using Adobe Photoshop 6 and Adobe Illustrator 10 and quantitative analysis of acquired images was performed by using ImageJ (http://imagej.nih.gov/ij/ ).
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