For the analysis of muscle fiber classification, Adenosinetriphosphatase (ATPase)-staining was performed on serial cryocut cross-sections (7μm) as previously described [47 (link)]. Muscle fiber phenotypes were matched in MyHC type I and type II fibers including IIA and IIX fibers. For muscle fiber type distribution, a mean total of 325 ± 125 muscle fibers were analysed for each participant, taken from sections at different depths of the muscle. The mean fiber type-specific diameter was determined on ATPase-stained sections using Scion Image (NIH, Bethesda, MD) calculating the ellipse minor axis [48 (link)]. To determine the number of myonuclei inside the muscle fiber, serial cryocut cross-sections (7μm) were stained with Mayer’s haematoxylin and counterstained with eosin. The cross-sections were examined under a Zeiss Observer microscope (Jena, Germany). The muscle fiber composition and the number of nuclei per muscle fiber (average of 141 fibers, range 80–162 fibers) were investigated using the PALM Robo V4 image analysis software (Zeiss, Jena, Germany). The mean cross-sectional area was determined in the same fibers using Scion Image (NIH, Bethesda, MD). For the calculation of the length of myonuclei, a range of 260–325 nuclei were quantified from serial sections from different depths of the vastus lateralis. Analyses were performed for each participant.
Scion image
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Scion Image is a digital image analysis software designed for scientific image processing and measurement. It provides tools for capturing, analyzing, and quantifying images from various scientific instruments and microscopes.
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Lab products found in correlation
Slot blot apparatus, by Bio-Rad (4 mentions)
Sis analysis program, by Olympus (4 mentions)
Ecl advanced detection system, by GE Healthcare (3 mentions)
α sma, by Merck Group (3 mentions)
Infrared ir labeled secondary antibodies, by LI COR (2 mentions)
Odyssey infrared imaging system, by LI COR (2 mentions)
Bcl 2, by Cell Signaling Technology (2 mentions)
Fibronectin, by Abcam (2 mentions)
Horseradish peroxidase conjugated secondary antibodies p447, by Agilent Technologies (2 mentions)
Collagen 1, by Abcam (2 mentions)
E cadherin, by BD (2 mentions)
Enhanced chemiluminescence reagent, by Thermo Fisher Scientific (2 mentions)
Bradford method, by Bio-Rad (2 mentions)
Nitrocellulose membrane, by Bio-Rad (2 mentions)
Primary antibody anti dinitrophenylhydrazone primary or anti protein bound hne, by Merck Group (2 mentions)
Tris glycine gel, by Thermo Fisher Scientific (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Vz 9000, by Keyence (1 mentions)
Statistica, by StatSoft (1 mentions)
Complete protease and phosphatase inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
91 protocols using scion image
1
Muscle Fiber Characterization Protocols
2
Western Blot Analysis of AH Proteins
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After determining the protein concentration, each AH sample (containing 4 mg of total AH protein) was mixed with 5× sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer and denatured at 100°C for 5 minutes. Then, the samples were separated by 15% gradient acrylamide SDS-PAGE. The separated protein bands were transferred onto a polyvinylidene difluoride (PVDF) blotting membrane (Millipore, Burlington, MA, USA) and incubated with primary rabbit anti-human antibodies for IL-16R (1:1000; ab2573; Abcam, Cambridge, MA, USA), IL-6 (1:1000; ab151538; Abcam), and β-actin (1:1000; ab8226; Abcam) at 4°C overnight. The membranes were then incubated with a goat anti-rabbit IgG horseradish peroxidase (HRP)-conjugated secondary antibody (1:1000; ab6721; Abcam) for 30 minutes at room temperature. The immunoblotted bands were visualized using an enhanced chemiluminescence reagent (Pierce ECL Western Blotting Substrate; Thermo Fisher Scientific). The gray band values of the blots were analyzed using Scion Image (Version: Beta 4.0.2 of Scion Image, Scion Corporation, Frederick, MD, USA).
3
Quantification of Cerebellar GFP Expression
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Cerebellar sections were stained for GFP and analyzed via Scion Image (Scion Corporation, Frederick, MD). Six evenly spaced sections per animal were analyzed. The GFP fluorescent immunoreactivity (Supplementary Figure S1A ) in the cerebellar vermis was captured at ×5 or ×2.5 magnification and converted to grayscale (Supplementary Figure S1B ). The Density Slice option in Scion Image was used to highlight the fluorescent pixels (Supplementary Figure S1C ). Only highlighted pixels were counted. The GFP signal that was quantified was highly specific and only found in AAV GFP transduced samples. Nontransduced tissues produced negligible background readings in this analysis. The average for six sections per animal was taken.
4
Digital Occlusal Cast Analysis
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Digital occlusal photographs were taken of all maxillary and mandibular casts according to a standard technique and imported into Adobe Photoshop, version 5.0 (Adobe Systems, San Jose, CA). Each photograph was saved with a 5-mm grid and then imported into Scion Image (Scion, Frederick, MD; a version of the Macintosh program, NIH Image, from the National Institutes of Health). Each point in Scion was recorded to an x, y coordinate system and imported into a Microsoft Excel program to orient the coordinates and align the maxillary and mandibular casts. The dental casts were measured at T0 and T1 for each patient.
5
SDS-PAGE and Western Blot Analysis of CaL Channel
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According to our previous report [28 (link)], the protein sample of cerebral artery was run for SDS-PAGE for 80 min at 30 mA with a 8% Tris-Glycine gel (Invitrogen, Carlsbad, California, USA). After separation, proteins were transferred onto a nitrocellulose membrane and then blocked overnight at 4 °C. The membranes were incubated with rabbit polyclonal antibody against the CaL channel α1C-subunit (Alomone Labs, Jerusalem, Israel) and subsequently incubated with Infrared (IR)-labeled secondary antibodies (LI-COR). A monoclonal mouse antibody raised against β-actin (Sigma) was used as a lane-loading control. The bound antibody was detected by the Odyssey infrared imaging system (LI-COR). Densitometry analysis of bands was performed by Scion image (Scion, Frederick, MD).
6
Quantitative Image Analysis of In Situ Hybridization
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In situ hybridization autoradiographs were digitized using Scion Image (Scion Corporation) in a single session per experiment to prevent image capture session artifacts during analysis. An individual blind to treatment group assignments used ImageJ (NIH) to measure average uncalibrated optical density (OD) values for regions of interest (Fig 1 ) corresponding to brain regions defined in the rat brain atlas by Paxinos and Watson [19 ]. For each region of interest, measurements were performed on both hemispheres of 4–6 sections per brain and then averaged.
7
Laser-Assisted Extraction Socket Evaluation
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Fig. 1 shows a schematic diagram of the laser irradiation site after tooth extraction. The number of α-SMA positive cells was measured in the area of granulation tissue formation on the surface of the extraction socket or the lamina propria in the tissue collected from the same site. The number of α-SMA-positive cells was measured within the scope of 150 μm in the vertical and horizontal directions at three locations with clear staining. The ratio of TGF-β1-positive areas per unit area was first calculated and then the average value of each group was calculated accordingly. A digital microscope (VZ-9000; KEYENCE CO., Ltd., Osaka, Japan) was used for the measurement. After scanning the image, a measurement was performed using Scion-image (Scion Corporation, Frederick, MD, USA). The evaluation was conducted by one co-author in a blind test. The measured values were shown as mean ± standard deviation, and a STATISTICAlly significant difference was determined by Fisher's exact test (STATISTICA; StatSoft, Tulsa, OK, USA) with a significance level of less than 5% (p < 0.05).Figure 1 ![]()
Morphological measurement methods. The measurement area was 150 × 150 μm in length and width, respectively, in the superficial granulation tissue formation or the intrinsic mucosal layer of the extraction socket of the first molar (M1: first molar, M2: second molar).
8
Slot Blot Assay for Oxidative Biomarkers
9
Quantitative Immunoblotting of dLGN Samples
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Mice were deeply anesthetized using Avertin (2,2,2-Tribromoethanol, Sigma-Aldrich), and brains were dissected into ice-cold PBS. Three hundred-micrometer coronal brain sections were then prepared using a McIlwain tissue chopper (Ted Pella, Inc.), and dLGN tissue samples were obtained by manual dissection using small scalpel blades. The tissue samples were flash frozen in liquid nitrogen or directly homogenized in lysis buffer (150 mM NaCl, 10 mM KCl, 20 mM HEPES, pH 7.0, 1 mM MgCl2, 20% glycerol, and 1% Triton X-100, including the complete protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific)). Twelve micrograms of protein per sample were separated by gel electrophoresis. After transfer and incubation with primary antibodies, HRP-conjugated secondary antibodies in combination with the ECL Prime Western blotting detection reagent (GE Healthcare Life Sciences) were used. Chemiluminescent signals were detected by exposure to photographic film (Kodak BioMax MR) and quantified by densitometry (Scion Image; Scion Corporation).
10
Quantifying Dopaminergic Fibers and Neurons
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The density of TH-positive fibers in the striatum of rats was determined and analyzed with a computerized analysis system as described previously [21] (link). Five sections at 0.5±1.0 mm anterior to the bregma were randomly selected for quantitative analyses [18] . The two areas adjacent to the needle tract of lesioned side and the symmetrical areas in the contralateral side were analyzed, respectively. The percentages of lesion to the intact side were evaluated in each section and the averages were used for statistical analyses. The images were computer-processed into binary images using an appropriate threshold (Scion Image, Scion Corp., Frederick, MD, USA). The areas were then calculated and used for statistical analyses. According to our previous publication for counting the number of TH-positive neurons [22] (link), every fifth 40 µm-thick coronal section through the substantia nigra pars compacta (SNc) was explored using 3 coronal sections, respectively, at 4.8, 5.3, and 5.8 mm posterior to the bregma. The number of cells was summed up in each group. The percentage to the intact side was analyzed and the average was used for the statistical analyses.
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