COCs were collected at 10 h post-hCG and denuded with 50 μL hyaluronidase (1000 IU/mL, Invitrogen Australia, Mulgrave, Vic, AU) in collection media (αMEM-HEPES). Denuded oocytes were washed in fresh collection media then incubated in media containing 6 μM JC-1 dye (GIBCO, Invitrogen Australia). Oocytes were incubated for 15 min at 37 °C to allow JC-1 aggregates to form in high-membrane potential mitochondria (red fluorescence). Oocytes were then immediately imaged through an optical cross-section at the widest point of the oocyte using the Fluoview FV10i Olympus confocal microscope for green fluorescence emission (525 nm, low membrane potential) and red fluorescence (590 nm, high membrane potential) using a 60× objective. A total of 58 oocytes from 4 KO mice and 61 oocytes from 4 heterozygous littermates were imaged (PRlacZ strain). Using AnalySIS LS professional software (Olympus Australia, Mt Waverly, Vic, AU), a rectangle was placed across the oocyte image and the average green or red fluorescence intensity in each pixel column (0.3 μm intervals) across the box was determined. The mean and SE of the red or green fluorescence was calculated across all PRKO or PGR heterozygous (PRHet) oocytes. Statistical significance was determined by a mixed-model two-way ANOVA.
Fluoview fv10i confocal microscope
Manufactured by Olympus
Sourced in Japan, United States, Germany
The Fluoview FV10i is a confocal microscope designed for high-resolution imaging. It utilizes laser-scanning technology to capture detailed images of fluorescently labeled samples. The microscope is equipped with multiple laser lines and sensitive detectors to enable the visualization of a variety of fluorescent probes. The FV10i is a compact, all-in-one system that provides researchers with a tool for advanced microscopy applications.
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Lab products found in correlation
Vectashield mounting medium, by Vector Laboratories (5 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (5 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (5 mentions)
Texas red x phalloidin, by Thermo Fisher Scientific (4 mentions)
Prolong gold, by Thermo Fisher Scientific (4 mentions)
Fv10 asw software, by Olympus (4 mentions)
Triton x 100, by Merck Group (4 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (4 mentions)
Fluoview image analysis software, by Olympus (4 mentions)
4 6 diamidino 2 phenylindole (dapi), by Roche (3 mentions)
Fluoview software, by Olympus (3 mentions)
Anti stat1 antibody, by Cell Signaling Technology (3 mentions)
8 well chamber slide, by Thermo Fisher Scientific (3 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (3 mentions)
Alexa fluor conjugated secondary antibody, by Thermo Fisher Scientific (3 mentions)
4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (3 mentions)
Syto9, by Thermo Fisher Scientific (3 mentions)
Dylight 488, by Thermo Fisher Scientific (3 mentions)
Anti mouse alexa 488 secondary antibodies, by Cell Signaling Technology (2 mentions)
Fv10 asw 4.0 viewer, by Olympus (2 mentions)
168 protocols using fluoview fv10i confocal microscope
1
Mitochondrial Membrane Potential Profiling
2
Mitochondrial Dynamics Visualization
3
Transdermal Dye Delivery Efficiency
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To visualize the efficiency of the normal and test fabrics on transdermal drug delivery, the topical application of a lipophilic dye, rhodamine B, was performed on the back skin of all group of each the guinea pigs. A confocal laser scanning microscope (Leica SP5 white laser; Leica Microsystems GmbH; magnification, x100) was then used to examine the dye delivery associated with each fabric. Rhodamine B base dye (0.0005 M; Sigma-Aldrich; Merck KGaA) was left to penetrate the skin for 3 h. Immediately after treatment, skin samples were collected and then washed with PBS to remove any residual rhodamine B and embedded in material at the optimal cutting temperature. Fixed skin tissue was frozen by immersion in liquid N2-cooled hexane and stored at -80˚C. Transverse sections (60 µm), including the whole right and left ventricles were obtained using a cryostat (Leica CM1325; Leica Microsystems GmbH) and mounted on glass. A DAPI mounting medium kit (OriGene Technologies, Inc.) was used to counterstain the nuclei for 10 min at room temperature, and stained cells were visualized using an Olympus FLUOVIEW FV10i confocal microscope (Olympus Optical Co., Ltd.; magnification x100).
4
NK-CdM Treatment Enhances Collagen in NHDFs
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NHDFs were seeded into a chamber slide, serum starved for 24 h, and then treated with NK-CdM for 48 h. Following treatment with 4% paraformaldehyde for 10 min at 4°C and 0.1% Triton X-100 for 5 min, the cultured NHDFs were incubated with an anti-type I collagen antibody (1:500; cat. no. ab34710; Abcam) at 4°C overnight and then with fluorescein isothiocyanate-labeled goat anti-rabbit IgG (1:1,000; cat. no. NB7182; Novus Biologicals, LLC). A 4′6-diamidino-2-phenylindole mounting medium kit (OriGene Technologies, Inc.) was used to counterstain the nuclei for 10 min at room temperature, and stained cells were visualized using an Olympus FLUOVIEW FV10i confocal microscope (Olympus Optical Co., Ltd.).
5
Fluorescent Imaging of PH(1-110)GFP Particles
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SF9 cells infected with recombinant baculoviruses, 72 hpi were washed with PBS and incubated for 5 min with DAPI (4′, 6-diamino-2-phenylindole) to mark the nucleus (Thermo Fisher, USA, cat. no. D3571) at a 1:1000 dilution and fixed in slide glass (76 × 26 mm) with DAKO Fluorescent Mounting Medium (Agilent, USA, cat. no. S3023) [28 (link)]. The GFP of the PH(1–110)GFP particles was excited at 473 and DAPI was excited at 405 nm. Fluorescence emission was collected at 510 nm for GFP and 420 nm for DAPI. All images were taken with a Fluoview FV10i confocal microscope (Olympus®, Japan), using the 60 × NA 1.35 oil immersion objective (UPLSAPO60XO). The images were analyzed with the software, FV10ASW.
6
PPAR-γ Expression in Ac2F Cells and Adipocytes
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The Ac2F cells and adipocytes (after differentiation) were seeded into the confocal dishes overnight. The cells were treated with parecoxib (15, 30, and 60 μM) and rosiglitazone (10 μM) for 48 h. Then, the cells were fixed with formalin for 10 min. The permeabilization of cells was achieved by incubating the cells with 0.3% Triton X-100 for 15 min. Next, the cells were blocked using 10% FBS and incubated with a PPAR-γ antibody at 4°C overnight. The cells were incubated with anti-mouse Alexa 488 secondary antibodies (Cell Signaling Technology) for 30 min, and then PI (10 μg/mL) and RNase (10 μg/mL) reagents were added to the cells and incubated for 40 min. Finally, FluoView FV10i confocal microscope (Olympus, Tokyo, Japan) was used to analyze the stained cells.
7
Immunofluorescence Visualization of Epithelial-Mesenchymal Transition
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Cultured NCI-H322M cells grown on coverslips in a 24-well plate were fixed with 2% paraformaldehyde (Nacalai Tesque, Kyoto, Japan) for 30 min, washed with PBS, and permeabilized with 0.1% Triton-X-100 (Thermo Fisher Scientific) in PBS for 15 min. Following a PBS wash, non-specific proteins were blocked with 2% BSA (Fujifilm Wako Pure Chemical) for 15 min. The cells were incubated with a mixture of two primary antibodies: anti-E-cadherin rabbit monoclonal antibody (1:200 dilution, 24E10; Cell Signaling Technology) and anti-N-cadherin mouse monoclonal antibody (1:200 dilution, 610920; BD Biosciences) for 1 h at RT. Coverslips were washed with PBS and incubated with CF®488A goat anti-rabbit IgG (1:200 dilution, Biotium, Hayward, CA, USA) and CF®568 goat anti-mouse IgG (1:200 dilution, Biotium) antibodies in the dark for 15 min. Following a PBS wash, nuclei were stained with Hoechst 33342 (5 μg/mL; Nacalai Tesque) for 15 min. The cells were then washed and mounted in an aqueous-based mounting medium, ClearMount™ (Thermo Fisher Scientific). Images were captured with a 60 × oil objective lens on an Olympus Fluoview FV10i confocal microscope (Olympus, Tokyo, Japan).
8
Immunofluorescence Staining of Cultured Cells
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After treatment, cells were fixed with 4% paraformaldehyde (PFA) for 20‐30 minutes at room temperature. The cells were permeabilized with 0.1% Triton X‐100 for 15 minutes. The cells were blocked in 0.1% bovine serum albumin (BSA) for 30 minutes and then incubated with primary antibody at 4°C overnight. After washing 3 times for 5 minutes with a TBS‐T solution, Alexa Fluor‐conjugated secondary antibodies (1:400) were used for visualization. Cell nuclei were stained with DAPI for 3 minutes. After washing 3 times for 5 minutes, the dish was covered with PBS. The images were collected on an Olympus FluoView FV10i confocal microscope.
9
Immunofluorescence Microscopy Protocol
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For immunostaining, cells were cultivated on slides and fixed with 3% formaldehyde for 10 min at room temperature. Immunostaining was performed as recently published by our group [22 (link)]. A Fluoview FV10i confocal microscope from Olympus was used for fluorescence microscopy. All measurements and image processing were performed using the company’s software. Optical magnification settings of 10X and 60X with oil were used. A laser with wavelengths of 647 nm (ALEXA 647), 594 nm (ALEXA 594) and 358 nm (DAPI) was selected for imaging. Laser power was manually optimized and used with equal settings for all imaged samples.
10
Immunostaining and Confocal Microscopy
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For immunostaining, cells were grown on slides and fixed with 3% formaldehyde for 10 min at room temperature. Afterwards the slides were incubated in permeabilization solution (HEPES, Sucrose, NaCl, MgCl2, 0.5% Triton X-100) for 20 min at 4 °C and subsequently blocked in PBS 2% BSA for 30 min at 37 °C. The slides were then washed three times for five minutes with PBS. The primary antibody was diluted in blocking buffer (2% BSA, PBS) and incubated for 90 min at 37 °C. After 3 washes (5 min) in PBS the secondary antibodies were diluted in blocking buffer and added to the coverslips for 45 min at 37 °C. After another 3 wash cycles a counterstaining with DAPI for 20 min followed. After another wash in water the coverslips were fixed on a glass plate. A Fluoview FV10i confocal microscope from Olympus was used for fluorescence microscopy. All measurements and image processing were performed using the company’s software. Optical magnification settings of ×10 and ×60 with oil were used. Laser power was manually optimized and used with equal settings for all imaged samples.
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