Cells on a cover glass (0117520; Paul Marienfeld, Lauda-Königshofen, Germany) were fixed with cold methanol for 10 min at 4℃, washed with cold PBS, and blocked with blocking solution (3% bovine serum albumin, and 0.3% Triton X-100 in PBS) for 30 min. The samples were incubated with primary antibodies for 1 h, washed with 0.1% PBST, incubated with secondary antibodies for 30 minutes, washed, and treated with 4,6-diamidino-2-phenylindole (DAPI) solution for up to 2 minutes. The cover glasses were mounted on a slide glass with ProLong Gold antifade reagent (P36930; Life Technologies). Images were observed with fluorescence microscopes with a digital camera (Olympus IX51) equipped with QImaging QICAM Fast 1394 and processed in ImagePro 5.0 (Media Cybernetics). ImagePro 5.0 (Media Cybernetics), Photoshop CC (Adobe) and ImageJ 1.51k (National Institutes of Health) were used for image processing. For measuring fluorescence intensities at centrosome, all images were obtained in an identical setting with the same exposure time. ImageJ was used for measuring and the background signals were subtracted from the centrosomal signals.
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Lab products found in correlation
Masson s trichrome staining, by Junsei (4 mentions)
Toluidine blue o, by Daejung Chemicals (4 mentions)
Microtome, by Leica Microsystems (4 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (4 mentions)
Sigmaplot, by Grafiti LLC (3 mentions)
Pdgf aa, by R&D Systems (2 mentions)
Mtt assay, by Merck Group (2 mentions)
Crystal violet, by Merck Group (2 mentions)
Microtome, by Leica camera (2 mentions)
Masson s trichrome, by Media Cybernetics (1 mentions)
Sc 376764, by Santa Cruz Biotechnology (1 mentions)
Vegfr2, by Cell Signaling Technology (1 mentions)
Carl confocal laser scanning microscope lsm 880, by Zeiss (1 mentions)
H2dcfda, by Thermo Fisher Scientific (1 mentions)
Ix83 microscope, by Olympus (1 mentions)
Prolong gold, by Thermo Fisher Scientific (1 mentions)
Evolution qei, by Media Cybernetics (1 mentions)
Axiovert 200, by Zeiss (1 mentions)
Sdf 1, by R&D Systems (1 mentions)
5 aza, by Merck Group (1 mentions)
41 protocols using image pro 5
1
Immunofluorescence Microscopy Protocol for Centrosome
2
Multivariate Characterization of Mesenchymal Stem Cells
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Several in vitro assays were performed to assess the cellular activities of MSCs. An MTT assay (Sigma-Aldrich) was used to assess cell proliferation, and a colony forming unit-fibroblast (CFU-F) assay was used to assess self-renewal. Multipotency (in vitro differentiation into chondrogenic, osteogenic, or adipogenic lineages) and transwell migration in response to platelet-derived growth factor (PDGF; 10 ng/mL PDGF-AA, R&D Systems, Minneapolis, MN, USA) were also assessed. Angiogenesis was quantified using Matrigel, and in vitro anti-inflammation was analyzed as described previously8 (link)–10 (link),20 (link),21 (link). The digital images generated in these assays were assessed quantitatively using Image-Pro 5.0 software (Media Cybernetics, Rockville, MD, USA).
3
Comprehensive Histological Analysis of Bladder Tissue
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Immediately after the awake cystometry, the bladder tissue was harvested for histological analysis, which evaluated the epithelial denudation, mast cell infiltration, tissue fibrosis, and apoptosis with cytokeratin immunostaining (Keratin, Pan Ab-1; Thermo Scientific, Foster City, CA, USA), toluidine blue staining (Toluidine blue-O; Daejung Chemicals and Metals, Seoul, Korea), Masson’s trichrome staining (Junsei Chemical, Tokyo, Japan), and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining (Roche, Mannheim, Germany), respectively [6 (link),7 (link),16 (link)]. Engraftment of the administered M-MSCs was determined by immunofluorescence analysis (EVOS, FL Color Imaging System, Life Technologies, Carlsbad, CA, USA) of human β2-microglobulin (hB2M) (mouse monoclonal, SC80668; Santa Cruz Biotechnology Inc., Paso Robles, CA, USA) and visualized by Alexa 488-conjugated (A11029) anti-mouse antibody (Molecular Probes, Grand Island, NY, USA), as previously described [2 (link),9 (link)]. The nuclei were counterstained with 4′,6′-diamino-2-phenylindole. Quantitative digital image analysis was performed in three randomly selected representative areas of each slide from five independent animals using Image-Pro 5.0 software (Media Cybernetics, Rockville, MD, USA).
4
Quantifying Bone Regeneration in Cranium
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Each cranium was dehydrated through a graded alcohol series ranging from 70% to 100%, and then embedded in polymethylmethacrylate. After hardening, longitudinal sections were cut into 150–200 μm slices using a microtome (Leica Microsystems Ltd, Wetzlar, Germany), glued onto a plastic support and then polished to a final thickness of approximately 50 μm. New bone formation and mineralization were quantified at four locations that equally divided the defect site between the two ends of the longitudinal sections. The mean value of the four measurements was calculated to give average values for each group. The sections were then stained with van Gieson’s picrofuchsin to evaluate new bone formation43 (link). The area of new bone formation was quantitatively evaluated at six random sections using Image Pro 5.0 software (Media Cybernetics, Rockville, MD, USA).
5
Histomorphometric Analysis of Bone Regeneration
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The bone specimens harvested at 8 and 16 weeks postoperatively were used for histological and histomorphometric analyses. Half of the specimens were fixed in 4% paraformaldehyde for 2 days. Then, the specimens were decalcified in 20% ethylenediaminetetraacetic acid for 30 days and dehydrated, embedded in paraffin, and sectioned. Sections were stained with HE and AM for the evaluation of bone structures and blood vessels as previously described39 (link). Meanwhile, the other half of the specimens was dehydrated in a graded alcohol series and then embedded in polymethylmethacrylate. After hardening, longitudinal sections were cut into 150–200 μm slices using a microtome (Leica), glued onto a plastic support, and then polished to a final thickness of approximately 50 μm. First, the sections were examined for fluorescent labeling. Then, the sections were stained with VG picrofuchsin to evaluate new bone formation. The area of new bone formation was quantitatively evaluated in four random sections using Image-Pro 5.0 software (Media Cybernetics).
6
Histological Analysis of Bladder Tissue
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After 24 hours of fixation in 4% paraformaldehyde, each bladder was embedded in paraffin and cut into 3-μm-thick slices that were affixed to slides and stained with hematoxylin and eosin. Mast cell infiltration and fibrosis were assessed via Toluidine Blue staining (Toluidine Blue-O, Daejung Chemicals & Metals Co., Seoul, Korea) and Masson’s Trichrome staining (Junsei Chemical, Tokyo, Japan), respectively. To assess apoptosis, bladder sections were stained with antibodies specific for terminal dUTP nick-end labeling (TUNEL) (Roche, Mannheim, Germany), followed by visualization using Alexa488-conjugated anti-mouse or rabbit antibodies (Molecular Probes, Grand Island, NY, USA). Quantitative digital image analyses were performed on seven randomly chosen representative areas of each slide. To quantify fibrosis and apoptosis, the areas staining with Masson’s Trichrome and the TUNEL reagent were calculated using Image Pro 5.0 software (Media-Cybernetics, Rockville, MD, USA). Mast cell infiltration was quantified by counting cells that stained with Toluidine Blue.
7
Bladder Histological and Genetic Analysis
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After awake cystometry, the bladder was harvested for histological and genetic expression analysis. Histological analysis was performed to evaluate epithelial denudation, vessels, mast cell infiltration, tissue fibrosis, and apoptosis with cytokeratin immunostaining (Keratin, Pan Ab-1; Thermo Scientific, MA, USA), CD31 staining (sc-376764; Santa Cruz Biotechnology, TX, USA), toluidine blue staining (Toluidine blue-O; Daejung Chemicals & Metals, Seoul, Korea), Masson’s trichrome staining (Junsei Chemical, Tokyo, Japan), VEGFR2 (#2472, Cell Signaling Technology, Danvers, MA, USA) and PDGFR-α (sc-398246; Santa Cruz Biotechnology, Dallas, TX, USA) respectively. Quantitative digital image analysis was performed in two randomly selected representative areas of each slide from five independent animals with Image-Pro 5.0 software (Media Cybernetics, Rockville, MD, USA). The PDGF and VEGF associated genes were quantified by real-time quantitative polymerase chain reaction (RQ-PCR) analysis10 (link). The overall shematic descripton of the experiment is graphically summarized in Supplementary Fig. 1 .
8
Quantitative Evaluation of Bone Formation
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One part of each cranium was dehydrated in a graded alcohol series ranging from 70% to 100%, and then embedded in polymethylmethacrylate. After hardening, longitudinal sections were cut into 150–200 μm slices using a microtome (Leica Microsystems Ltd, Wetzlar, Germany), glued onto a plastic support and then polished to a final thickness of approximately 50 μm. First, the sections were examined using a CLSM (Leica, Heidelberg, Germany) for fluorescent labeling. Then, new bone formation and mineralization were quantified at four locations that equally divided the defect site between the two ends of the longitudinal sections. The mean value of the four measurements was calculated to give average values for each group. The sections were then stained with van Gieson’s picrofuchsin to evaluate new bone formation6 (link). The area of new bone formation was quantitatively evaluated at four random sections using Image Pro 5.0 software (Media Cybernetics, Rockville, MD, USA).
9
Biomechanical Evaluation of Prefabricated Bone Grafts
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Four months after bone graft prefabrication, compression tests were performed on six prefabricated bone grafts from the periosteal flap group, six prefabricated bone grafts from the muscular pouch group, eight native cancellous bone samples from the iliac crest, and eight native cortical bone samples from the radius to evaluate the biomechanical properties. These samples were chosen as controls because of their similar cross-sectional area and the fact that both bone qualities (cancellous and cortical bone) were used for bone defect reconstruction. Prior to mechanical testing, the test samples were cut into cylinders with the same diameter as the prefabricated bone grafts (4 mm) and a height of 8 mm. Then, the cross-sectional area and height of the test samples were measured using Image-Pro 5.0 software (Media Cybernetics). The samples were tested under uniaxial compression using a biomechanical analyzer (Instron-8874, Canton, MA) according to a previously described protocol39 (link).
10
Callose and ROS Detection in Plant Roots and Leaves
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A pot experiment was conducted as described above. Twenty roots and mature leaves were sampled for callose and reactive oxygen species (ROS) detection at 24 h post-inoculation. This experiment was conducted with three independent biological replicates.
Callose deposition was detected using the aniline blue staining method53 (link). The stained leaves and roots were visualized using an Olympus IX83 microscope with a UV filter (Olympus Corporation, Tokyo, Japan; excitation filter 350/50 nm, replicated DM 400 dichroic beam splitter and emission filter 460/50 nm). ImagePro 5.0 software (Media Cybernetics, Bethesda, MD) was used to convert the colored micrographs to binary, and pixel densitometry was performed across the 20 images from each treatment.
ROS production was examined with the oxidant-sensitive probe dichlorodihydrofluorescein diacetate (H2DCFDA; Invitrogen, California, USA)54 (link). The samples were imaged with a Carl Zeiss confocal laser scanning microscope (LSM 880; Carl Zeiss, Oberkochen, Germany).
Callose deposition was detected using the aniline blue staining method53 (link). The stained leaves and roots were visualized using an Olympus IX83 microscope with a UV filter (Olympus Corporation, Tokyo, Japan; excitation filter 350/50 nm, replicated DM 400 dichroic beam splitter and emission filter 460/50 nm). ImagePro 5.0 software (Media Cybernetics, Bethesda, MD) was used to convert the colored micrographs to binary, and pixel densitometry was performed across the 20 images from each treatment.
ROS production was examined with the oxidant-sensitive probe dichlorodihydrofluorescein diacetate (H2DCFDA; Invitrogen, California, USA)54 (link). The samples were imaged with a Carl Zeiss confocal laser scanning microscope (LSM 880; Carl Zeiss, Oberkochen, Germany).
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