Immunostained astrocytes were imaged using an Olympus Scanning Confocal Microscope (FV1000) with a 60x oil objective (1.35 NA), at a 10 μs/pixel scanning speed and Kalman averaging value of 2. For each astrocyte, z-stack images were taken at 0.5 μm step size to capture TG2 and Zbtb7a signal throughout all planes of the nucleus. Approximately 5 astrocytes per coverslip were imaged. In Imaris, images were deconvoluted and max intensity projections were created. Before the colocalization of Zbtb7a and TG2 signals were quantified, the entire image except for any nuclei were masked. Once only nuclear signal remained the background subtraction feature was used in the TG2 channel to minimize non-specific signal. Puncta with a diameter less than 3μm were filtered out. The co-localization function in Imaris was used to determine the proportion of each signal overlapping with the other. Within the co-localization function intensity thresholds were set at 320 for TG2 and 260 for Zbtb7a. Co-localization values of each channel were reported as Mander’s coefficients.
Fv1000 scanning confocal microscope
Manufactured by Olympus
Sourced in Japan
The FV1000 is a scanning confocal microscope designed for high-resolution imaging. It utilizes laser-based illumination and a pinhole aperture to capture optical sections of a specimen, allowing for the visualization of three-dimensional structures. The FV1000 is capable of providing detailed, high-quality images, making it a versatile tool for various scientific and research applications.
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Lab products found in correlation
Plapon, by Olympus (3 mentions)
Tricaine, by Merck Group (2 mentions)
C apochromat, by Zeiss (2 mentions)
Fitc dextran, by Merck Group (2 mentions)
Tissue tek oct compound, by Sakura Finetek (2 mentions)
Alexa 488, by Thermo Fisher Scientific (2 mentions)
Tunel assay kit, by Roche (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Alexa 555, by Thermo Fisher Scientific (2 mentions)
Bcl2 associated x (bax), by Santa Cruz Biotechnology (2 mentions)
Bcl 2, by Santa Cruz Biotechnology (2 mentions)
Fluoroshield mounting medium with dapi, by Merck Group (1 mentions)
Paraformaldehyde (pfa), by Electron Microscopy Sciences (1 mentions)
Goat anti mouse alexa 488 secondary antibody, by Thermo Fisher Scientific (1 mentions)
Saponine, by Merck Group (1 mentions)
Lysotracker red dnd 99, by Thermo Fisher Scientific (1 mentions)
Lab tek, by Thermo Fisher Scientific (1 mentions)
Fv1000, by Olympus (1 mentions)
Vectashield, by Vector Laboratories (1 mentions)
Fluoview 3, by Olympus (1 mentions)
32 protocols using fv1000 scanning confocal microscope
1
Quantifying Astrocytic Zbtb7a and TG2 Colocalization
2
Quantifying Astrocyte Zbtb7a and TG2 Colocalization
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Immunostained astrocytes were imaged using an Olympus Scanning Confocal Microscope (FV1000) with a 60× oil objective (1.35 NA), at a 10 μs/pixel scanning speed and Kalman averaging value of 2. For each astrocyte, z-stack images were taken at 0.5 μm step size to capture TG2 and Zbtb7a signal throughout all planes of the nucleus. Approximately five astrocytes per coverslip were imaged. In Imaris, images were deconvoluted and max intensity projections were created. Before the colocalization of Zbtb7a and TG2 signals were quantified, the entire image except for any nuclei was masked. Once only the nuclear signal remained the background subtraction feature was used in the TG2 channel to minimize non-specific signal. Puncta with a diameter of less than 3 μm were filtered out. The co-localization function in Imaris was used to determine the proportion of each signal overlapping with the other. Within the co-localization function intensity thresholds were set at 320 for TG2 and 260 for Zbtb7a. Co-localization values of each channel were reported as Mander’s coefficients.
3
BrdU Incorporation and Imaging
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Normal cultured cells were incubated with 10 μmol/L BrdU for 60 min. After fixing with 4% paraformaldehyde (PFA) and permeabilizing with 0.2% Triton X-100/phosphate-buffered saline (PBS) (V/V) for 10 min, cells were treated with 2 mol/L HCl for 30 min. After that, cells were incubated with rat anti-BrdU primary antibody overnight at 4 °C, followed by goat anti-rat cyanine3 conjugated secondary antibody for 60 min. Slides were mounted with Permount (Polysciences, USA, Cat. No. 18606-20) and an Olympus scanning confocal microscope (FV1000, Japan) was used to capture the images.
4
Telomere Dysfunction and DNA Damage
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Cultured cells were fixed with 4% PFA for 10 min, washed with PBS, and dehydrated in successive 70%, 85%, and 100% ethanol treatments (5 min each). Hybridization buffer (20 mmol/L Na2HPO4; 20 mmol/L Tris; 60% formamide; 2×saline sodium citrate (SSC, 0.3 mol/L NaCl, 30 mmol/L Na citrate, pH=7.4)) containing 0.05 pmol/L (T2AG3)-Cy3-labeled peptide nucleic acid (PNA) telomeric probe was added to the cells, followed by heating to 85 °C for 2 min. Cells were placed into a dark humidified chamber at 37 °C overnight. After 4% PFA fixation, the cells were re-stained with anti-γH2A.X or anti-EPAS1 antibodies following standard immunocytofluorescence protocols. DAPI staining was performed to label the nuclei. Slides were analyzed using an Olympus scanning confocal microscope (FV1000, Japan). For TIF analysis, three independent experiments (n=100 cells each) were performed by counting the co-localization of telomeric foci and γH2A.X. For telomere FISH-based quantification of telomere length, 20 cells were examined by measuring the integrated density of telomeric foci using ImageJ software (v1.8.0).
5
Zebrafish Cardiac Immunofluorescence Imaging
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Zebrafish immunofluorescence staining images were taken using an Olympus FV1000 scanning confocal microscope. The dissected embryo hearts were mounted in 1% low-melt agarose. The confocal images were captured with an UPLSAPO 40×. Immunostained cell images were collected using a Leika with an UPLSAPO 60X objective.
6
Dox-Loaded Sphere Uptake in Cells
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SKBR3 and MSU1.1 cells (1 × 105 cells/well) were plated on 8-well Lab-Tek chambered coverslips (Nunc, Naperville, IL) and cultured for 24 h. Next, 10 μg/mL of Dox-loaded H2.1MS1:MS2, H2.1MS1:DOXMS2, and MS1:DOXMS2 spheres were added to the cells, which were incubated at 37 °C for 15 or 30 min. After washing with PBS, the cells were fixed with 4% paraformaldehyde (PFA; Electron Microscopy Sciences, Hatfield, PA). Subsequently, the cells were washed with PBS and immersed in Fluoroshield mounting medium with DAPI (Sigma, St. Louis, MO) and then analyzed under an Olympus FV1000 scanning confocal microscope (Shinjuku, Tokyo, Japan) connected to a blue laser diode and an argon laser. Image acquisition and analysis were performed with a 60× objective, a 1.4 N.A. oil immersion lens, and FLUOVIEW Viewer software, ver. 4.1. The nuclei were visualized using 350 nm excitation and 440–480 nm emission wavelengths. To visualize the Dox-loaded spheres and the Dox released from the spheres, an excitation wavelength of 488 nm and an emission wavelength of 570–610 nm were used.
7
Immunostaining of Embryonic Zebrafish Hearts
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Hearts were harvested manually from the embryos at 52 hpf and fixed with 4% paraformaldehyde (PFA) in PBS overnight at 4°C, followed by the permeabilization with PBS containing 0.1% Tween-20 and 0.5% Triton-X 100 for 10 min. Then the hearts were blocked in blocking buffer with 1% BSA and 10% normal goat serum for 3 h at room temperature. Mouse anti-Alcam antibody (Developmental Studies Hybridoma Bank, United States) was added in the blocking buffer and incubated for 16 h at 4°C. Goat anti-mouse Alexa-488 secondary antibody (Thermo Fisher Scientific, United States) was added in blocking buffer after thorough washing and incubated overnight at 4°C. After washing with PBS, all the samples were stained with 100 nM Tetramethylrhodamine (TRITC) labeled phalloidin solution (Solarbio, Beijing, China) containing 1% BSA. AVC cells were recognized based on their characteristic cuboidal morphologies. Images were taken using an Olympus FV1000 scanning confocal microscope. The confocal images were captured with an UPLSAPO 40 × objective.
8
Neuromuscular Junction Quantification
9
Immunofluorescence Assay for Lipid Signaling
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Forty thousand cells were seeded on non-fluorescent gelatin-coated coverslips and allowed to adhere for 30 min. Following stimulations, as indicated in figure legends, cells were fixed with 2% paraformaldehyde in PBS for 10 min at room temperature. Where indicated, cells were permeabilized with 0.05% saponine (Sigma-Aldrich, St. Louis, MO, USA) in PBS for 20 min and blocked with 2% BSA in PBS for 30 min. Then, cells were incubated with the appropriate primary antibodies for 2 h, and secondary antibodies for 1h or fluorescent phalloidin for 45 min, as indicated within the Figure legends. Images were taken with a FV1000 scanning confocal microscope (Olympus, Tokyo, Japan) coupled to an inverted microscope using a 63× oil immersion objective. For quantification of ATX/LPP or ATX/LPAR1 co-localization, cells were incubated with ATX and LPP1, LPP2, LPP3, or LPAR1 antibodies. The percentage of co-localization was calculated as previously described from serial optical sections of the whole cell [78 (link)].
10
Visualizing PCSK9 Trafficking and Lysosomal Colocalization
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SV-589 cells were cultured on Lab-Tek (Nunc) 8-well chambered coverglass and incubated overnight in sterol-depleting Medium C containing 150 μM E64. For colocalization of Alexa488-labeled PCSK9 with LysoTracker Red DND-99 (Life Technologies) cells were labeled with Alexa488-labeled wild-type PCSK9 (30 µg/ml) or D347Y PCSK9 (5 µg/ml) for 1 h and chased for up to 6 h in label-free Medium C containing E64. LysoTracker Red DND-99 was incubated at a concentration 200 nM for 2 h prior to the end of each chase period. For colocalization studies of Alexa488-labeled PCSK9 and Alexa647-labeled transferrin, cells were preincubated for a minimum 1 h in serum-free Medium A to deplete endogenous transferrin. Cells were then incubated with Alexa488-labeled wild-type PCSK9 (30 µg/ml) or PCSK9-D347Y (5 µg/ml), and Alexa647-labeled transferrin (100 µg/ml) for 1 h in Medium A. The labeled proteins were chased for 2 h in label-free Medium A containing E64, then visualized directly. Images were taken on an Olympus FV1000 scanning confocal microscope. Colocalization was quantified using Image J (http://rsb.info.nih.gov/ij/ ).
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