Bimolecular fluorescence complementation (BiFC) assays was performed as follows. Leaves of N. benthamiana were inoculated with A. tumefaciens strains containing expression vector pNPP40-nGFP-NbRanBP1-1a and pNPP40-cGFP-Ran1a, or pNPP40-nGFP-NbRan1a and pNPP40-cGFP-RanBP1-1a. Fluorescence images for GFP were recorded ˜2 days after inoculation under a FV1000-D microscope (Olympus) as described above.
Fv1000 d microscope
Manufactured by Olympus
Sourced in Japan
The FV1000-D is a high-performance confocal laser scanning microscope designed for advanced imaging applications. It features a fully motorized, high-precision optical system and a compact, modular design. The FV1000-D provides exceptional image quality, resolution, and sensitivity, making it suitable for a wide range of research and analysis tasks.
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Lab products found in correlation
Ab45179, by Abcam (3 mentions)
Ab133273, by Abcam (3 mentions)
Ab201687, by Abcam (3 mentions)
In cell analyzer 6000, by GE Healthcare (1 mentions)
Ab112134, by Abcam (1 mentions)
Mcsp24h48, by Merck Group (1 mentions)
Picogreen, by Thermo Fisher Scientific (1 mentions)
Tetramethylrhodamine ethyl ester, by Thermo Fisher Scientific (1 mentions)
Cellmatrix 1 a, by Nitta Gelatin (1 mentions)
Scs n08, by Matsunami (1 mentions)
Anti iba1, by Fujifilm (1 mentions)
Protein block, by Agilent Technologies (1 mentions)
Biotin conjugated anti cd11b ab, by BioLegend (1 mentions)
Perfecthyb plus hybridization buffer, by Merck Group (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Glass coverslip, by Thermo Fisher Scientific (1 mentions)
Attofluor cell chamber, by Thermo Fisher Scientific (1 mentions)
14 protocols using fv1000 d microscope
1
Bimolecular Fluorescence Complementation Assay
2
Mitochondrial membrane potential analysis
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Mitochondrial membrane potential assay was performed using the cationic dye JC10, and an assay kit (Abcam, ab112134), following the manufacturer’s protocol. Confocal images of JC10 stained cells were acquired on same settings using Olympus FV1000D microscope (Olympus, Tokyo, Japan). Following image acquisition, fluorescence intensity of JC10 aggregates and monomers were analyzed using ImageJ software. For the assessment of JC10 staining of extracellular mitochondria, cells were seeded onto the membrane of 24-well hanging culture inserts (Merck Millipore, MCSP24H48) with lower compartment well coated with poly-L-lysine. On the following day, cells were subjected to DMSO or rotenone treatment for 4 h. Inserts containing cell monolayers were removed from the well compartment and the extracellular particles were allowed to sediment by mild centrifugation at 2000 × g for 10 min before JC10 staining and microscopic observation using IN Cell Analyzer 6000 (GE healthcare, Chicago, IL, USA). Sixty-four images covering the entire area of culture well were taken at 20× magnification. Automated quantification of red and green fluorescently labeled mitochondria was performed using the software IN Cell Developer Toolbox.
3
Evaluating Mitochondrial Dysfunction and Autophagy
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Adult mouse cardiomyocytes on laminin-coated glass-based dishes (IWAKI Cell Biology, Bio-REV Pte. Ltd., Singapore) were incubated with 100 nmol/l carbonyl cyanide m-chlorophenyl hydrazine (CCCP) for 6 h. To estimate mitochondrial membrane potential, the cells were treated with 10 nmol/l of tetramethylrhodamine ethyl ester (Molecular Probes, Eugene, Oregon) for 30 min. To visualize DNA and autophagosomes, the cells were incubated in three-dimensional gel with Cellmatrix I-A (Nitta Gelatin Inc., Osaka, Japan) and fixed with methanol at −30°C for 15 min. The cells were incubated with anti–microtubule-associated protein light chain (LC) 3B antibody (Cell Signaling Technology, Danvers, Massachusetts) overnight at 4°C, followed by staining with anti-rabbit Alexa 568 secondary antibody (Abcam, Cambridge, United Kingdom) overnight. The cells were incubated with 100-fold diluted PicoGreen (Thermo Fisher Scientific) for 30 min before confocal microscopic analysis using an FV-1000D microscope (Olympus, Tokyo, Japan) (7) (link).
4
Immunostaining Procedure for Cellular Proteins
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Immunostaining was performed as described before [31 (link)]. Briefly, cells were grown on cover glasses and fixed for 15 min with 4% paraformaldehyde (PFA) solution at room temperature. Cells were washed twice with PBS and then permeabilized with 0.1% Triton-X100 in PBS for 10 min. Then, cells were incubated with a blocking buffer (PBS with 5% BSA). Primary antibody was applied at the specified dilution in blocking solution overnight at 4 °C. The following morning, cells were washed twice with PBS and incubated with the appropriate fluorescent secondary antibody (in blocking solution) for 30 min in the dark. Coverslips were then washed three times with PBS, mounted with DAPI, and examined under Olympus FV1000D microscope. The following antibodies were used for immunostaining: ARHGEF2 (ab201687, Abcam), CHGA (ab45179, Abcam), and AR (ab133273, Abcam).
5
Immunofluorescence Analysis of AHNAK in Dental Pulp
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DP-1 and priDPC cells were seeded into 8-well non-coated glass chamber slides (SCS-N08; Matsunami, Osaka, Japan), fixed with 3.7% formaldehyde for 10 min, permeabilized with 0.5% Triton X-100 in DPBS for 5 min, blocked with 1% BSA in DPBS for 1 h at room temperature, and incubated with anti-AHNAK antibody (1:200 dilution) overnight at 4°C. On the following day, the cells were incubated with Cy3-conjugated goat anti-rabbit secondary antibody (1:1000 dilution) for 1 h at room temperature and embedded with SlowFade™ Diamond Antifade Mountant containing DAPI (Life Technologies). Immunofluorescence signals were observed using an FV1000-D microscope (Olympus). Human dental pulp tissues dissected from healthy teeth extracted for orthodontic purposes were fixed with 3.7% formaldehyde for 24 h, sequentially immersed in 15% sucrose in PBS overnight and 30% sucrose overnight, embedded in OCT compound, and sectioned at 10-μm thickness. Rehydrated sections were treated as described above to detect AHNAK expression in human dental pulp tissues.
6
Analyzing Microglial Subtypes in GFP Mice
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LN− cells that were isolated from GFP mice and cultured on astrocytes were fixed in 4% paraformaldehyde. After treatment with protein block (Dako, Denmark), samples were immunostained with: (1) biotin-conjugated anti-CD11b Ab (BioLegend, San Diego, CA, USA) followed by DyLight 649-Streptavidin (Jackson ImmunoResearch, West Grove, PA, USA), (2) anti-TREM2 Ab (R&D systems) followed by rhodamine-anti-sheep IgG (Jackson ImmunoResearch), (3) anti-CX3CR1 (R&D systems) followed by Cy5-anti-goat IgG (Jackson ImmunoResearch), or (4) anti-Iba1 (Wako Pure Chemical, Osaka, Japan) followed by rhodamine-anti-rabbit IgG Ab (Jackson ImmunoResearch). Images were acquired using an FV1000-D microscope (Olympus, Tokyo, Japan). To quantify the number of cells, ten fields under a ×20 objective were randomly selected, and GFP-positive or immunostained cells were counted using ImageJ software (National Institutes of Health, Bethesda, MD, USA). SR cells and LF cells were defined as cells with a circularity of >0.8 and ≤0.8, respectively.
7
Immunostaining Procedure for Cellular Proteins
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Immunostaining was performed as described before [31 (link)]. Briefly, cells were grown on cover glasses and fixed for 15 min with 4% paraformaldehyde (PFA) solution at room temperature. Cells were washed twice with PBS and then permeabilized with 0.1% Triton-X100 in PBS for 10 min. Then, cells were incubated with a blocking buffer (PBS with 5% BSA). Primary antibody was applied at the specified dilution in blocking solution overnight at 4 °C. The following morning, cells were washed twice with PBS and incubated with the appropriate fluorescent secondary antibody (in blocking solution) for 30 min in the dark. Coverslips were then washed three times with PBS, mounted with DAPI, and examined under Olympus FV1000D microscope. The following antibodies were used for immunostaining: ARHGEF2 (ab201687, Abcam), CHGA (ab45179, Abcam), and AR (ab133273, Abcam).
8
Confocal Microscopy Imaging Workflow
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Confocal laser-scanning fluorescence microscopy was performed with an FV1000-D microscope (IX81; Olympus), using an objective lens with a long working distance (LUCPLFLN 60×, NA 0.7). The image was obtained by the acquisition software FV10-ASW4.2. The contrast and the brightness of the obtained images were adjusted with Adobe Photoshop.
9
Fluorescence Imaging of Cellular Structures
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Fluorescence images were recorded between 425 and 475 nm (calcofluor white), between 495 and 520 nm (GFP) or between 570 and 590 nm (RFP) under a FV1000-D microscope (Olympus) using 405 nm (calcofluor white), 488 nm (GFP), or 559 nm (RFP) excitation.
10
CD82 Monoclonal Antibody Binding Validation
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The binding of human CD82 mAb (Santa Cruz Biotechnology) to the surface of EOL-1R cells was confirmed using an Olympus FV1000-D microscope (data not shown).
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