A Chameleon Ti:Sapphire laser (Coherent, Glasgow, UK) connected to a Chameleon compact opo system was controlled by Olympus Fluoview FV500 software (25 (link)). A 20× water immersion lens (0.95 NA) was used for in vivo and slice imaging. eGFP, 2,000 KDa dextran-FITC, 70 KDa dextran-Texas Red and yellow Hoechst were excited using a 900 nm laser. An 800 nm laser was tuned to 1280 nm by the opo system for 2DG-IR excitation. Bandpass filters (Chroma) were 540/40 for eGFP and FITC, 650/75 nm for yellow Hoechst, 675/65 nm for tdTomato and Texas Red and 855/210 nm for 2DG-IR emission. Single channel acquisition was used for 2DG-IR and dual channel acquisition was used for combination of other fluorophores.
Fluoview fv500
Manufactured by Olympus
Sourced in Japan, United States, United Kingdom
The Fluoview FV500 is a laser scanning confocal microscope system designed for fluorescence imaging. It is capable of capturing high-resolution, three-dimensional images of biological samples.
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Lab products found in correlation
Cellquest pro software, by BD (5 mentions)
Lsm 700, by Zeiss (2 mentions)
Fluo 3 am, by Thermo Fisher Scientific (1 mentions)
Prolong gold antifade reagent with 4 6 diamidino 2 phenylindole dapi, by Thermo Fisher Scientific (1 mentions)
Ms excel, by GraphPad (1 mentions)
Cytation 5 microplate reader, by Agilent Technologies (1 mentions)
N cadherin, by Thermo Fisher Scientific (1 mentions)
Anti cytokeratin 19, by Abcam (1 mentions)
Fluoromount aqueous mounting medium, by Merck Group (1 mentions)
Antifade mounting medium, by Beyotime (1 mentions)
Transwell insert, by Corning (1 mentions)
Matrigel, by BD (1 mentions)
Acidic tyrode s solution, by Merck Group (1 mentions)
Facscalibur, by BD (1 mentions)
Culture slides, by BD (1 mentions)
Annexin 5 fitc detection kit, by BD (1 mentions)
Ultracruz hard set mounting medium, by Santa Cruz Biotechnology (1 mentions)
Fluoromount g, by Thermo Fisher Scientific (1 mentions)
Deadend fluorometric tunel system, by Promega (1 mentions)
Vectashield mounting media, by Vector Laboratories (1 mentions)
43 protocols using fluoview fv500
1
Multimodal Imaging with Chameleon Laser
2
Multimodal Imaging with Chameleon Laser
3
Quantifying Intracellular Calcium Dynamics
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The measurement of [Ca2+] I was performed, according to that described in our previous study (34 (link)). The acutely dissociated neurons and Mg2+-free treated hippocampal neurons were incubated for 20 min with 5 µm fluo-3 acetoxymethylester (fluo-3/AM; Molecular Probes Life Technologies, Carlsbad, CA, USA; 32°C; 5% CO2; pH adjusted to 7.4 with NaOH). The neurons of the control group and model group were plated into two cell culture cover glasses in the same dish. Thus, the control and model neurons were treated using the same procedure. The labeled neurons were then rinsed three times with PBS and cultured for 30 min to exclude non-specific staining of the extracellular fluid (32°C; 5% CO2). The preparations were observed and quantitatively analyzed using a confocal laser scanning biological microscope (Fluoview FV500; Olympus, Corporation). The visual field was selected where at least five neurons contained fluo-3/AM, which was excited by the 488 nm line of a 200 mW argon ion laser and captured at wavelengths >505 nm. The data were expressed as relative fluorescence intensities. Confocal imaging was performed on three separate fields of cells for each group. The concentration of intracellular Ca2+ was expressed as the relative fluorescent intensity using ImageJ 1.46 software (National Institutes of Health, Bethesda, MD, USA).
4
Apelin-13 Neuroprotection against Rotenone
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To assess the neuroprotective effects of apelin-13 against rotenone induced apoptosis, nuclear morphology was detected using a previously described method in our lab [77 (link),78 (link)]. SH-SY5Y cells were cultured on coverslips in 24-well plates. Cells were fixed in 4% paraformaldehyde (PFA) for 30 min, washed twice with PBS, and stained with Hoechst 33258 dye for 15 min at room temperature. After washing 3 times to remove the excess dye, cells were examined and photographed under a confocal laser scanning microscope (Fluoview FV500, Olympus, Osaka, Japan). Based on changes in nuclear morphology, such as chromatin condensation and fragmentation, apoptotic cells are defined. We delineated a 400 μm2 frame in each space, and then selected 10 different regions to calculate all condensed nuclei and normal nuclei. Then we calculated the average sum of the condensed nuclei and normal nuclei for each hole. The data were presented as percentages, and the proportion of condensed nuclei relative to the total number of nuclei was calculated.
5
Immunofluorescence Imaging of NOTCH1 and β-Catenin
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Cells were seeded onto 4-chamber slides before being fixed and permeabilized. The fixed cells were incubated with anti-NOTCH1and anti-β-catenin (all diluted 1:100) for 1 h at room temperature. The cells were subsequently incubated with the Cy3-conjugated goat anti-rat and FITC-conjugated goat anti-rabbit secondary antibodies diluted 1:100 for 30 min at room temperature. The cell nuclei were stained with ProLong Gold antifade reagent with 4′,6-diamidino-2-phenylindole (DAPI) (Invitrogen). Protein expression and localization were examined using a confocal microscopy system (FluoView FV500, Olympus).
6
Immunofluorescence Staining of EAC Cells
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The immunofluorescence staining was performed on OE19 and JHESO EAC cells treated with PVT1 ASO at 1 μM as performed as previously described [18 (link)]. Expression and localization of the proteins were observed under a confocal microscope system (FluoView FV500; Olympus) and analyzed by CellQuest PRO software (BD Biosciences, San Jose, CA). Information for antibodies used in immunofluorescent staining and western blot was described in Additional file 6 : Figure S6.
7
Dansylated PLLA Surface Analysis
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An Olympus FluoView FV500 confocal laser scanning with 20 times magnification (20×) was used to analyze the fluorescence of dansylated PLLA surfaces (for Dansyl λexcitation≈ 335 nm and λemission≈ 518 nm). Confocal fluorescence microscopy micrographs were taken without any previous special sample preparation.
8
Liposomal Uptake and Cytotoxicity in B16F10 Cells
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Uptake in B16F10 cells was evaluated from incubation with rhodamine-6G dye-loaded liposomes and imaging with confocal laser scanning microscopy (CLSM; Olympus Fluoview FV500, Olympus, Tokyo, Japan). Cells were grown on glass coverslips and incubated with the formulation and free dye for 3 h, after which the excess dye was washed away with PBS; then, the cells were fixed on the glass slides with 10% formalin, and imaged with CLSM. Average fluorescence intensities were calculated using ImageJ software. Cytotoxicity assay was done with liposome-PTX and LG-PTX on B16F10 cells for 72 h. Cell viability was measured (using sulphorhodamine-B assay) at increasing drug concentrations (10–10,000 nM), and IC50 was calculated using MS Excel and GraphPad.
9
Visualizing particle distribution in larvae
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Distribution of fluorescent 50 nm PS NPs alone, and of non-fluorescent 500 nm PS NPs and 4.5 µm MPs with sorbed B(a)P, within brine shrimp larvae and developing zebrafish embryos was examined under a confocal microscope (Olympus Fluoview FV500, Tokyo, Japan). All the individuals used for these analyses were exposed at the same time as those used for toxicity assays and at the same exposure concentrations. Transmitted and fluorescence confocal images of Z-stacks series were acquired using a 10x UPLAPO NA0.45 lens. Fluorescence images were obtained by excitation at 405 nm and emission at 430–460 nm. Fluorescence emitted by B(a)P accumulated in brine shrimp larvae and zebrafish embryos exposed to B(a)P alone was analyzed at the end of the toxicity assays using a Cytation 5 microplate reader (Biotek Instruments Inc., Winooski, VT, USA) provided with a blue filter (360/460 nm excitation/emission wavelengths). For imaging, brine shrimp larvae and zebrafish embryos were anesthetized with 2% (v/v) chloroform in salt water and with 200 mg/L benzocaine prepared in embryo water, respectively.
10
Immunohistochemical Analysis of Frozen Tumor Samples
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Serial cryostat sections from human and mice tumor samples, immediately snap frozen in OCT after removal, were stained with hematoxylin and eosin and processed for immunohistochemistry as described [3 (link)]. Cell counting was carried out in 8–12 randomly chosen fields independently by three researchers in a blind fashion; images were acquired with Leica DM RX microscopy using Scion Image software. Sections were stained with 1μM LysoSensor Green immediately after cutting. The specificity control was obtained by pre-incubating serial cryostat sections with buffer at pH 8.8 for 10 min before Lysosensor staining. Images were analyzed by confocal microscopy, acquired with Fluoview FV500 software (Olympus BioSystems) and the fluorescence was quantified using ImageJ software.
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