Fv1000 confocal microscope

Manufactured by Olympus

Sourced in Japan, United States, Germany, China, United Kingdom, France, Australia, Switzerland

The FV1000 confocal microscope is a high-performance imaging system designed for a wide range of applications in biological and materials research. It features advanced confocal technology, enabling high-resolution, optical sectioning of samples with minimized out-of-focus light. The FV1000 provides precise control over imaging parameters, allowing researchers to capture detailed, three-dimensional images of complex specimens.

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1 548 protocols using fv1000 confocal microscope

Investigating Protein Interactions in U251 Cells

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To investigate the interaction between Aβ and CAV2, TGFB2 or TGFBR1, U251 cells were inoculated with β-amyloid (1–42), HiLyte Fluor™ 488-labelled (ANASPEC, AS-60479-01). After 1 h, the cells were washed, fixed and incubated with CAV2 (Abcam, ab133484), TGFB2 (Abcam, ab36495) or TGFBR1 antibodies (Abcam, ab31013). The cells were washed, counterstained with DAPI and observed under an Olympus FV1000 confocal laser microscope. To study the interaction between NEAT1 and P300/CBP, U251 cells were incubated with the NEAT1 probe overnight at 37 °C and then the anti-P300 (Abcam, ab59240) or anti-CBP antibodies (Abcam, ab50702) for 1.5 h at room temperature. After the cells were washed and incubated with the secondary antibody, they were counterstained with DAPI and mounted for observation. Cell images were obtained with an Olympus FV1000 confocal microscope. To study the role of NEAT1 in the interaction between STAT3 and H3K27Ac, U251 cells were transfected with a NEAT1 or negative control siRNA for 36 h and then incubated with the anti-STAT3 Y705 (Abcam, ab76315) and anti-H3K27Ac antibodies (Abcam, ab4729) for 1.5 h at room temperature. After the cells were washed and incubated with the secondary antibody, they were counterstained with DAPI and mounted for observation. Cell images were obtained with an Olympus FV1000 confocal microscope.

Wang Z., Zhao Y., Xu N., Zhang S., Wang S., Mao Y., Zhu Y., Li B., Jiang Y., Tan Y., Xie W., Yang B.B, & Zhang Y. (2019). NEAT1 regulates neuroglial cell mediating Aβ clearance via the epigenetic regulation of endocytosis-related genes expression. Cellular and Molecular Life Sciences, 76(15), 3005-3018.

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Autophagy Visualization in Cancer Cells

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For autophagosome and autophagic flux examination, cancer cells were plated on cover sides at 20% confluence in 24-well plates. After the treatment was performed as needed, the cells were washed with ice-cold PBS and fixed with 4% paraformaldehyde for 30 min at room temperature. Then, the cells were blocked in a 1% BSA solution in PBS for 1 h at room temperature and washed with PBS supplemented with 1% BSA three times followed by 0.1 mg/mL DAPI (Cat# 9542, Sigma, USA) for 10 min in the dark. Finally, the slides were washed with PBS and covered with glycerin. Cells were imaged using an Olympus FV-1000 confocal microscope. The number of LC3B puncta per cell in GFP-positive or mCherry-GFP-positive cells was determined as reported previously^{47 (link)}.
For colocalization immunofluorescence assays, cells were seeded on cover sides. After the treatment was performed as needed, the slides were fixed with 4% paraformaldehyde, blocked with PBS supplemented with 1% BSA and then permeabilized with 0.1% Triton-100. Next, slides were incubated with the primary antibody at 4 °C overnight. Then, the slides were embedded with fluorochrome-conjugated secondary antibody and incubated with DAPI in the dark for 10 min. Representative images were obtained using an Olympus FV-1000 confocal microscope.

Hu F., Song D., Yan Y., Huang C., Shen C., Lan J., Chen Y., Liu A., Wu Q., Sun L., Xu F., Hu F., Chen L., Luo X., Feng Y., Huang S., Hu J, & Wang G. (2021). IL-6 regulates autophagy and chemotherapy resistance by promoting BECN1 phosphorylation. Nature Communications, 12, 3651.

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Visualizing Zinc in BCG-Infected Cells

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For visualization of free zinc, the THP-1 cells infected with DiI-labeled M. bovis BCG strains were incubated for 1 h with the cell permeant reagent FZ3 (Invitrogen) at a final concentration of 1 mM in PBS. The confocal images were acquired using the Olympus FV1000 confocal microscope. An UPlanSApo100x/1.40 NA oil objective was used to obtain an over sampled 1024 × 1024 image with 49 nm pixel and a z-stack with a step size of 120 nm, recorded with Zen software (Carl Zeiss, Inc.), and analyzed with Olympus FV1000 confocal microscope.

Chen L., Li X., Xu P, & He Z.G. (2022). A Novel Zinc Exporter CtpG Enhances Resistance to Zinc Toxicity and Survival in Mycobacterium bovis. Microbiology Spectrum, 10(2), e01456-21.

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Monitoring Mitochondrial-Lysosomal Dynamics

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SH-SY5Y cells grown in a 35-mm glass bottom plate (MatTek, P35G-1.5-14-C) were co-transfected with GFP-LC3 and CellLight Mitochondria-RFP (Invitrogen, C10505). At 24 h after transfection, cell images of GFP-LC3 and Mito-RFP were captured with an Olympus FV1000 confocal microscope. To quantify the colocalization between GFP-LC3 and Mito-RFP, more than 100 cells expressing both Mito-RFP and GFP-LC3 were calculated according to the number of mitochondria-localized LC3 puncta.
To determine the colocalization between mitochondria and lysosomes, SH-SY5Y cells were loaded with 200 nM MitoTracker Green FM (Invitrogen, M7514) and 25 nM LysoTracker Red DND-99 (Invitrogen, L-7528) for 20 min at 37°C. Cell images were obtained with an Olympus FV1000 confocal microscope. The colocalization between mitochondria and lysosomes was calculated according to the number of mitochondria-localized lysosomes.

Cheng M., Liu L., Lao Y., Liao W., Liao M., Luo X., Wu J., Xie W., Zhang Y, & Xu N. (2016). MicroRNA-181a suppresses parkin-mediated mitophagy and sensitizes neuroblastoma cells to mitochondrial uncoupler-induced apoptosis. Oncotarget, 7(27), 42274-42287.

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Immunohistochemistry of Tubulin Modifications

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Sectioning and immunohistochemistry were performed using previously described protocols (Malicki et al., 2011 (link)). Following fixation, transverse cryosections, 20 μm thick, were taken through the retina or the ear and processed for staining with the following antibodies: anti-acetylated a-tubulin (rabbit) (D20G3; 1:1000; Cell signaling) Acetyl-α-Tubulin (Lys40) (mouse) (6-11B-1), anti-polyglutaminated tubulin GT335 (1:500; Adipogen) anti-monoglycylated tubulin TAP952 (1:500; Millipore) (Alexa Fluor 488- and 564-conjugated secondary antibodies were used for all staining procedures (1:1000) as well as DAPI for counterstaining. Images of cryosections were collected at the Wolfson Light Microscopy Facility using an Olympus FV1000 confocal microscope and a 60x immersion objective. Images of immunostained wholemount embryos were collected using the Olympus FV1000 confocal microscope with a 40× dipping lens.

Łysyganicz P.K., Pooranachandran N., Liu X., Adamson K.I., Zielonka K., Elworthy S., van Eeden F.J., Grierson A.J, & Malicki J.J. (2021). Loss of Deacetylation Enzymes Hdac6 and Sirt2 Promotes Acetylation of Cytoplasmic Tubulin, but Suppresses Axonemal Acetylation in Zebrafish Cilia. Frontiers in Cell and Developmental Biology, 9, 676214.

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Mitochondria-Lysosome Colocalization Assay

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C2C12 myoblasts stably transfected with Ad-GFP-LC3 (Hanbio, Shanghai, China) were seeded on glass cover slips. Twenty-four hours after transfection, the cells were treated with or without APN (30 μg/mL up to 24 h). The cells were then stimulated with H₂O₂ for 30 min. After that, cells were fixed for 15 minutes with 4% paraformaldehyde and washed twice with PBS 1X. Cells were blocked and permeabilized with PBS 1X + 0.2% Triton X-100 for 15 minutes at room temperature. After washing twice with PBS 1X, cells were incubated with a rabbit monoclonal TOMM20 antibody (Abcam) diluted 1:250 in 5% BSA O/N at 4 °C and washed twice with PBS 1X followed by incubation with a secondary anti-rabbit IgG antibody, conjugated to Alexa 555 (1:200) for 1 hour at room temperature. Coverslips were washed twice with PBS 1X and mounted on glass slides with fluorescent mounting medium Fluoroshield™ with DAPI (eBioscience) and visualized in an Olympus FV1000 confocal microscope.
To determine colocalization between mitochondria and lysosomes, C2C12 myoblasts were loaded with 200 nM MitoTracker Green FM (Beyotime, C1048) and 50 nM LysoTracker Red (Beyotime, C1046) for 30 min at 37 °C. Cell images were obtained using an Olympus FV1000 confocal microscope. The colocalization of mitochondria and lysosomes was analyzed according to the number of mitochondria-localized lysosomes.

Ren Y., Li Y., Yan J., Ma M., Zhou D., Xue Z., Zhang Z., Liu H., Yang H., Jia L., Zhang L., Zhang Q., Mu S., Zhang R, & Da Y. (2017). Adiponectin modulates oxidative stress-induced mitophagy and protects C2C12 myoblasts against apoptosis. Scientific Reports, 7, 3209.

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Visualizing PAK Isoform Localization and Cell-Surface Interactions

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Plasmids coding for fluorescently labeled PAK isoforms were prepared as described previously [29 (link)]. MOLM-7 cells (6 × 10⁵) were transfected using the Neon^TM transfection system (1325 V, 10 ms, 3 cycles) and 0.5 µg of DNA. The transfected cells were cultured for 24 h in RPMI medium without antibiotics, incubated for 1 h on fibronectin-coated coverslips, and localization of PAK1-full and PAK2 was analyzed by fluorescence microscopy (FV-1000 confocal microscope, Olympus).
For analysis of changes in the cell-surface contact area, cells were incubated for 90 min on fibronectin-coated surface, treated for 30 min with 10 µM FRAX597, and analyzed in the interference reflection mode. The measurement was performed by means of FV-1000 confocal microscope (Olympus), using the 405 nm laser beam and focusing on the glass surface.

Kuželová K., Obr A., Röselová P., Grebeňová D., Otevřelová P., Brodská B, & Holoubek A. (2021). Group I p21-activated kinases in leukemia cell adhesion to fibronectin. Cell Adhesion & Migration, 15(1), 18-36.

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Melanoma Cell Binding Assay

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B16/F10 and M21 melanoma cells (1 × 10⁵ cells per well) were seeded in a 4-well Lab-Tek Chamber Glass Slide System from Thermo Scientificand incubated at 37° overnight. After 24 h, the cells were fixed with 4% PFA in PBS and incubated at room temperature for 15 min, washed with PBS 3 times, treated with 0.5% Triton X-100 at room temperature for 15 mins and washed with PBS for 3 times. The cells were incubated with 1 μM of Cy5.5-GGNle-CyCMSH_hex with/without 1 μM of NDP-MSH peptide blockade at room temperature for 1 h and washed with PBS 3 times. Then the cells were stained for nuclei and mounted with DAPI Fluoromount-G mounting medium from SouthernBiotechand stayed in dark at room temperature for 24 h. The fluorescent signal was observed and recorded at 100 × magnification under an Olympus FV1000 confocal microscope.
Paraffin embedded B16/F10 and M21 melanoma sections (5 μm thickness) were incubated with 1 μM of Cy5.5-GGNle-CyCMSH_hex with/without 1 μM of NDP-MSH peptide blockade at room temperature for 1 h after deparaffinization with xylene. Tissue samples were then washed with PBS three times and stained and mounted with Prolong Diamond anti-fade mounting reagent with DAPI from Life Technologies. The fluorescent signal was observed and recorded at 100 × magnification under an Olympus FV1000 confocal microscope.

Yang J., Xu J., Gonzalez R., Lindner T., Kratochwil C, & Miao Y. (2018). 68Ga-DOTA-GGNle-CycMSHhex targets the melanocortin-1 receptor for melanoma imaging. Science translational medicine, 10(466), eaau4445.

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Prevascularized Constructs Immunofluorescence Analysis

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The co-cultured prevascularized constructs were washed with PBS, fixed and stained with mouse polyclonal antibody against human CD31 (Abcam, Cambridge, MA), and followed by DyLight 488 horse anti-mouse IgG secondary antibody (Vector laboratories, Burlingame, CA). The samples were washed and incubated in 4′, 6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich) solution to label the cell nuclei. Images were captured under an Olympus FV-1000 confocal microscope. The vascular networks were quantified by the ImageJ software, using methods previously described 43 . We firstly chose one vessel that aligned along the main direction of the nanogratings and set the angle of it as 0 degree, the angles of other vessels were measured and defined as Δ Angle. Experiments were triplicated, and 5 non-overlapping panels were randomly analyzed from each individual sample for unbiased statistical analysis. The same immunofluorescent staining was performed with anti-CD146 and anti-CD166 primary antibody staining. CD146 is known as the melanoma cell adhesion molecule (MCAM), which is regarded as a pericyte marker 44 (link). CD166 stains activated leukocyte cell adhesion molecule, and it was used to detect hMSC-EC interaction 45 (link). Images were captured under an Olympus FV-1000 confocal microscope by depth scan every 1 µm. The obtained images were reconstructed into 3-D views by a Fiji software.

Qian Z., Sharma D., Jia W., Radke D., Kamp T, & Zhao F. (2019). Engineering stem cell cardiac patch with microvascular features representative of native myocardium. Theranostics, 9(8), 2143-2157.

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Evaluating Cytotoxicity of Plant Extracts

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After 10 days of culture, multicellular and single-cell spheroids were treated for 48 h with the calculated IC₅₀ and sublethal concentrations of the P2Et and Anamu-SC extracts and DX (1/5 IC₅₀). As a negative control, extract diluent (ethanol) and DMSO for DX were used. Spheroids were collected and centrifuged at 300× g for 30 s and resuspended in 100 µL of LIVE/DEAD™ Cell Imaging Kit (for green fluorescence in live cells Ex/Em: 488–515 nm; for red fluorescence in dead cells Ex/Em: 570/602 nm; Invitrogen^®, Waltham, MA, USA) to assess cell viability through the FV1000 confocal microscope (Olympus^®, Tokyo, Japan). Using the Olympus FV1000 confocal microscope, 640 × 640 images of 3 and 5 spheroids were acquired with a UPLFLN 10X/NA 0.3 objective and the 488 and 515 nm laser lines, and analyzed using FIJI analysis software (ImageJ, version 1.51^®). For cytotoxicity analysis, the area from single focal plane spheroids was delimited and pixels’ area for each fluorescence channel (calcein and ethidium homodimer) was calculated. The data were analyzed and graphed using GraphPad Prism 8 software (GraphPad Software, Inc^®, San Diego, CA, USA). Area from single focal plane spheroids was delimited.

Corzo Parada L., Urueña C., Leal-García E., Barreto A., Ballesteros-Ramírez R., Rodríguez-Pardo V, & Fiorentino S. (2023). Doxorubicin Activity Is Modulated by Traditional Herbal Extracts in a 2D and 3D Multicellular Sphere Model of Leukemia. Pharmaceutics, 15(6), 1690.

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