Immunohistochemistry was performed as described previously [35 (link)]. Tumor sections prepared from control and silibinin-treated groups were stained with Ki67 (Thermo Fisher Scientific, Waltham, MA, USA) and c-Myc (clone 9E10; Santa Cruz Biotechnology, Dallas, Texas, USA), pSTAT3 (Cell Signaling Technology, Danvers, MA, USA) and GLUT1 (Abcam, Cambridge, UK) primary antibodies. Images were captured at 20X magnification using an inverted microscope (Leica, DMI600B) and processed by using Leica LAS AF software.
Dmi600b
Manufactured by Leica
Sourced in Germany, Switzerland
The DMI600B is a high-performance inverted microscope designed for a wide range of microscopy applications. It features a stable and rigid frame that supports precise optical alignment and delivers consistent image quality.
Automatically generated - may contain errors
Lab products found in correlation
Las af software, by Leica (5 mentions)
Wga alexa fluor 488 conjugate, by Thermo Fisher Scientific (3 mentions)
Vt1200, by Leica (3 mentions)
C myc clone 9e10, by Santa Cruz Biotechnology (1 mentions)
P stat3, by Cell Signaling Technology (1 mentions)
Glut1, by Abcam (1 mentions)
Opti mem medium, by Thermo Fisher Scientific (1 mentions)
Application suite version 3, by Leica (1 mentions)
Dmi3000b, by Leica (1 mentions)
Jsm 7800f, by JEOL (1 mentions)
Alexa fluor conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Rat anti cd68, by Bio-Rad (1 mentions)
Orca flash 4, by Hamamatsu Photonics (1 mentions)
Csu w1, by Yokogawa (1 mentions)
Biocoat matrigel invasion chamber, by BD (1 mentions)
Calcimycin a23187, by Merck Group (1 mentions)
Poly l lysine coated, by Merck Group (1 mentions)
Infinitef50r, by Fujifilm (1 mentions)
Ethos 1, by Milestone (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
37 protocols using dmi600b
1
Immunohistochemical Analysis of Tumor Markers
2
Transfection of Mutant Calcium-Sensing Receptors
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Human embryonic kidney cells (HEK293) (ATCC) were cultured under standard condition (5% CO2, 37°C) in High Glucose Dulbecco's modified Eagle's medium (DMEM) (Sigma Chemicals) containing 10% fetal bovine serum with 100 µg/ml penicillin-streptomycin. The CaSR mutations were introduced using site-directed mutagenesis (QuikChange, Stratagene, La Jolla, CA, USA) [14] (link). Cells were transfected with either pEGFP-N1-WT-CaSR or mutant CaSRs using Lipofectamine 2000 in reduced serum Opti-MEM medium following the manufacturer's instructions (Invitrogen). After 4∼6 hours, the medium was changed to High Glucose DMEM, and the cells were incubated for an additional 48 hours to increase receptor expression. Transfection efficiency and expression levels were confirmed by analyzing the fluorescence intensity of the EGFP-tagged CaSR by fluorescence microscopy (Leica, DMI600B).
3
Tracking Hippocampal Progenitor Dynamics
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Primary hippocampal progenitors were plated on poly-D-lysine and laminin coated 24 well plates, and cultured for 5 days. At 5 DIV, cultures were imaged on an inverted Leica DMI600B microscope, at 37 °C, with 5% CO2 in air. Cells were imaged for 2 h, with image acquisition every 5 min. Total distance covered was determined through manual cell tracking using the MTrackJ plugin in ImageJ52 . A minimum of 50 cells ware analysed per condition per animal.
4
Histological Analysis of Atherosclerotic Lesions
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En face and histological assessment of atherosclerotic lesion size has been described (11) (link). For histological aortic root analysis, frozen 5-μm sections from the aortic valve plane in 50-μm intervals were stained with Oil Red O staining with hematoxylin and light green counterstain. Picrosirius red stain and Masson’s trichrome stain with hematoxylin and eosin counterstain were used for assessment of fibrosis. For immunofluorescence, polyclonal anti-mouse CD3 (Dako A0452), anti-mouse B220 (RA3-6B2), anti-mouse CD11b (M1/70), anti-mouse CD11c (N418), polyclonal anti-mouse α-smooth muscle actin (ab15734) (Abcam, Cambridge, United Kingdom, and BioLegend), and the following secondary antibodies were used: donkey–anti-rat–IgG–AF488 (Invitrogen, Carlsbad, California), goat–anti-hamster–Cy3 (Jackson ImmunoResearch, Newmarket, United Kingdom), and donkey–anti-rabbit–IgG-AF555 (Life Technologies A31572, Life Technologies, Carlsbad, California). Images were obtained with a Leica DMI600B or DMI3000B microscope with 5×, 10×, and 20× original magnification using Leica Application Suite version 3.5.0 (Leica, Wetzlar, Germany). Analysis was conducted with NIH ImageJ and GIMP (version 2.8).
5
Calcium Imaging of Fura-2-Loaded Cells
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Cultures were loaded for 35–40 min at 37°C with 2 μM Fura-2-AM in Krebs-Ringer solution buffered with HEPES, 125 mM NaCl, 5 mM KCl, 1.2 mM MgSO4, 2 mM CaCl2, 10 mM glucose, and 25 mM HEPES (pH 7.4) and were washed twice with prewarmed Krebs-Ringer solution before recordings were made. The recording setting comprised an inverted microscope (Leica, DMI600B) equipped with a Ca2+ imaging unit. Polychrome IV (TILL Photonics; Germany) was used as a light source. Fura-2 fluorescence images were collected with a Andor CCD Camera (Axon Instruments, CA, USA) and analyzed with Imaging WorkBench 6 (INDEC BioSystem, Santa Clara USA). Single-cell 340/380 nm fluorescence ratios were analysed with Origin 6.0 (Microcal Software Inc., MA, USA).
6
Quantifying RAD51 and BRCA1 Foci in Skin Cells
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Fixed cells were imaged at room temperature using a CSU-X spinning disc confocal microscope (Intelligent Imaging Innovations) on an inverted microscope (DMI600B; Leica), with 63×, 1.4 NA objective lens and a charge-coupled device camera (cool-SNAP HQ2, Photometrics). Z stack images were obtained and saved in Slidebook software (Intelligent, Imaging, Innovations), where maximum intensity projections were created. For RAD51 and BRCA1 foci analysis in control skin fibroblast and Seckel cells, >150 cells for each sample were imaged and analyzed per replicate. The fraction of cells with more than 10 distinct RAD51 foci or more than 5 BRCA1 foci were determined for each replicate. The graph was plotted using the arithmetic mean and SEM derived from four independent biological replicates. A two-tailed Student's t test with 95% confidence interval was used for the statistical analysis.
7
GUS Staining of Transgenic Roots
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AM inoculated and non-inoculated transgenic roots carrying the SlPT4 promoter-GUS fusion were subjected to GUS staining based on a technique earlier developed by Jefferson [39 (link)]. Hairy roots were vacuum-infiltrated with a GUS staining solution composed of 0.05 M sodium phosphate buffer, 1 mM potassium ferrocyanide, 1 mM potassium ferricyanide, 0.05% Triton X-100, 10.6 mM EDTA-Na and 5 μg ml−1 X-gluc cyclohexylammonium salt (previously dissolved in N,N-dimethylformamide) for 30 min to improve substrate penetration. The tissues were then incubated in the dark at 37 °C from 1 h to overnight or until staining was satisfactory in the same staining solution. For co-staining of the AM fungus, GUS-stained hairy roots were embedded in hot (∼ 60 °C) liquid 4% agarose in PBS 1X. 60 μm longitudinal sections were cut on a vibratome (Leica VT1200S) and vacuum-infiltrated with 10 μg ml−1 WGA-Alexa Fluor 488 conjugate (Molecular Probes, Eugene, Oreg., USA) in PBS 1X for 20 min in the dark. Cuttings were washed with PBS 1X. A stereomicroscope Leica M165F and an inverted fluorescent microscope (Leica DMI600B) were used for visualization.
8
Osteoblast Morphology and Distribution
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In this work, osteoblasts were seeded at a density of 5 × 104 cells/cm2 onto the substrates which were placed in 24-well culture plates. After being cultured for 2 days, the samples were fixed with 4% paraformaldehyde and gently rinsed with PBS. For SEM observation, samples were dehydrated several times in gradient ethanol solutions, followed by dried treatment with t-butanol for 30 min. Cell morphology was observed by field emission scanning electron microscopy (FE-SEM; JSM-7800F, JEOL, Akishima, Japan). Then for cell fluorescence observation, the samples were permeabilized with 0.2% Triton X-100. Then Rhodamine-Phalloidin and Hoechst 33258 were used for staining of cell cytoskeleton and cell nuclei, respectively. Finally, fluorescence microscopy (Leica DMI 600B, Wetzlar, Germany) was employed to detect the morphology and distribution of cells.
9
Immunofluorescence analysis of γH2A.X
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S2-013 and T3M4 cells were seeded at ~ 40% density on sterile glass cover slips in 12 well plates. The next day cells were treated with indicated amount of silibinin for 48 h. After treatment cells were washed with PBS to remove the media and immunofluorescence staining was performed as described earlier [51 (link)] using phosphorylated H2A.X (γH2A.X) antibody (1:500 dilution). Images were captured at 20X magnification using an inverted microscope (DMI600B from Leica) and processed by using Leica LAS AF software.
10
Fluorescence Imaging of Allele-Specific Probes
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Samples were imaged on a Leica DMI600B automated wide-field fluorescence microscope equipped with a 100× Plan Apo objective, a Pixis 1024BR cooled charge-coupled device camera, and a Prior Lumen 220 light source. We imaged cells by taking a series of Z-stacks spaced by 0.35 µm. We tuned the exposure times depending on the dyes used: 2000 msec for the H19 guide probe, 4000 msec for the C6 and B6 allele-specific probes, and 4000 msec for the Atto488 probe.
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