The RAW 264.7 macrophage cells (5×105 cells/ml in each well) were incubated on glass coverslips and treated with PH-EPS (200 µg/ml) or LPS (1 µg/ml, for positive control) for 1 h. Subsequently, the cells were rinsed with cold PB at least three times and fixed with 4% paraformaldehyde at 37°C for 30–60 min. Thereafter, the cells were permeabilized with 0.2% Triton X-100, blocked with bovine serum albumin (5% in PBS; Gibco; Thermo Fisher Scientific, Inc.) at 37°C for 30 min, and incubated with anti-NF-κB p65 (1:100) at 37°C for 30 min. Then, the cells were thoroughly rinsed in PBS and incubated with a Cy3-labeled goat anti-rabbit IgG secondary antibody (cat. no. BA1032; Wuhan Boster Biological Technology, Ltd.) at 1:100 for 30 min at 37°C. DAPI was used as a nuclear counterstain at 37°C for 30 min. Images were captured via an Olympus FV300 confocal laser scanning microscope (Olympus Corporation).
Fv300 laser scanning confocal microscope
Manufactured by Olympus
Sourced in Japan, United States
The FV300 is a laser-scanning confocal microscope manufactured by Olympus. It is designed to provide high-resolution imaging of biological samples by using a laser to scan the sample and capture images. The FV300 allows users to visualize and analyze the structure and function of cells and tissues at the microscopic level.
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Lab products found in correlation
Lipofectamine 3000, by Thermo Fisher Scientific (5 mentions)
Fluoview software, by Olympus (4 mentions)
Hct116, by RIKEN Cell Bank (3 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Fluoview 300, by Olympus (2 mentions)
Bovine serum albumin (bsa), by Thermo Fisher Scientific (1 mentions)
Lab tek 2 chamber slide, by Thermo Fisher Scientific (1 mentions)
Anti chop, by Santa Cruz Biotechnology (1 mentions)
Alexa 488 and alexa 647 conjugated secondary antibodies, by Thermo Fisher Scientific (1 mentions)
Anti ubiquitin, by Santa Cruz Biotechnology (1 mentions)
Bodipy, by Thermo Fisher Scientific (1 mentions)
Nih3t3, by RIKEN Cell Bank (1 mentions)
Duolink in situ red starter kit mouse rabbit, by Merck Group (1 mentions)
Cho k1, by RIKEN Cell Bank (1 mentions)
Fluoview acquisition software, by Olympus (1 mentions)
Medium 199, by Thermo Fisher Scientific (1 mentions)
Kanamycin sulfate, by Merck Group (1 mentions)
Dmi6000 microscope, by Leica (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Pentr directional topo vector, by Thermo Fisher Scientific (1 mentions)
35 protocols using fv300 laser scanning confocal microscope
1
NF-κB Activation in RAW 264.7 Macrophages
2
FRAP Assay of mLRP4-GFP-HA in MIO-M1 Cells
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MIO-M1 cells transiently transfected with mLRP4-GFP-HA were seeded on Nunc Lab-Tek II chamber slides. After 24 h, the cells were incubated with vehicle or α2M* as previously described. The FRAP experiments were performed in an Olympus FV300 confocal laser scanning microscope equipped with a 60 × PLAPON oil immersion/1.42 NA (Olympus, Japan) objective, a 488 nm Argon laser, and a temperature controller set at 37 °C. Photo-bleaching was performed using 3× optical zoom and 300 scans of a region of interest (ROI) of 40 × 70 pixels covering a specific peripheral region of the cell at 8 µs/pixel, 100% transmission of 488 nm laser line. Pre- and post-bleaching images (5 and 100 images, respectively) were acquired every 2.7 s with a 512 pixel × 512 pixel resolution using the same objective. FRAP experiments were performed on at least 10 cells per condition and repeated experiments at least twice. Individual FRAP measurement curves were averaged to get a single FRAP curve. Data analysis was performed as previously published by Zheng and collaborators51 .
3
Immunocytochemistry and Immunohistochemistry Protocols
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For immunocytochemistry, cells grown on poly-L-lysine-coated coverslips were fixed with 4% paraformaldehyde for 10 min and permeabilized with 0.5% Triton X-100 in PBS for 10 min. After washing step with PBS, cells were incubated in a blocking solution (2% goat serum in PBS) and then incubated with primary antibodies. For immunohistochemistry, mouse brain sections were prepared according to the method previously described24 (link). The following primary antibodies were used: anti-p62 (CST), anti-ubiquitin (Santa Cruz), anti-αS (CST), anti-CHOP (Santa Cruz), anti-phospho-neurofilament (Covance), anti-phospho-c-Jun (CST), and anti-cleaved caspase-3 (CST). Positive immunostaining was detected using Alexa 488- and Alexa 647-conjugated secondary antibodies (Molecular Probes). Nuclei were counterstained with DRAQ7 (BioStatus) or TO-PRO3 iodide (Molecular Probes). Fluorescence images were analyzed with an FV300 confocal laser scanning microscope (Olympus).
4
FRET Analysis of Protein Interactions
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CHO-K1 cells were seeded onto 35-mm dishes containing a coverslip and grown to 70–80% confluence at the time of transfection, which was performed as indicated. Then cells were washed with PBS and fixed with 1% paraformaldehyde, and these fixed cells were washed with PBS and rinsed with Milli-Q water. Coverslips were visualized using an Olympus FV300 confocal laser scanning microscope. A Plan Apochromat 60×/1.42 NA oil immersion objective lens was used with digitally zoomed 3×, 512 × 512 image resolution. The ECFP (donor) and EYFP (acceptor) chimeric proteins were excited with an argon laser at 458 and 515 nm, respectively, and the emission channel was 465–495 nm for the donor and 530–560 nm for the acceptor. As a positive control, cells transfected with a chimeric CFP fused to YFP was used. Sensitized emission measurement was chosen, with images processed using the PFRET module for the FV10-ASW software of the FV1000 microscope.
5
Immunohistochemical Analysis of FVIII Expression
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Immunohistochemical analysis of FVIII expression in both untreated and vector-treated hemophilia A mice were performed as previously reported [11] , [12] with minor modifications. In brief, after tail-clip challenge tests, the mice were sacrificed, multiple organs liver, spleen, kidney, heart, and lung) were harvested, fixed with 10% formalin, embedded in paraffin and sectioned for staining. To assess transgene expression in tissues, specimens were stained with Cy3-labeled human anti-FVIII IgG [32] using the FluoroLink Ab labeling kit (Amersham Biosciences, Buckinghamshire, UK). Staining images were captured with FV300 confocal laser scanning microscope (Olympus Co., Tokyo, Japan).
6
NF-κB Translocation in RAW 264.7 Cells
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Twenty-four hours after seeding on glass coverslips, RAW 264.7 cells were washed with PBS. Then, cells were fixed immersing the coverslips in 4% neutral paraformaldehyde for 10 minutes, and 0.1% Triton-X100/PBS was added for 5 minutes at room temperature to improve permeability. The cells were blocked with 5% bovine serum albumin for 20 minutes and then incubated with NF-κB p65 antibody (1:100) for 1.5 hours to investigate the nuclear translocation of active NF-κB. Following incubation, the cells were washed with PBS and then stained for 1 hour with a fluorescein isothiocyanate-conjugated donkey anti-rabbit immunoglobulin G antibody (1:100). Nuclei were stained with 4′,6-diamidino-2-phenylindole (1:5,000; Thermo Fisher Scientific), and the cells were mounted and analyzed on an Olympus FV-300 confocal laser scanning microscope controlled by FLUOVIEW software (Olympus, Tokyo, Japan). The data were evaluated from three independent experiments in each group.
7
Adipocyte Lipid Droplet Imaging
8
Visualization of canine Ku80 localization
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The Madin–Darby canine kidney (MDCK) cell line (HSRRB, Osaka, Japan), the canine lung adenocarcinoma (CLAC) cell line (HSRRB), a mouse embryonic fibroblast cell line (NIH3T3; Riken Cell Bank, Tsukuba, Japan), a murine lung epithelial (Ku70+/−MLE) cell line, a human cervical carcinoma cell line (HeLa; Riken Cell Bank), and a human colon cancer cell line (HCT116; Riken Cell Bank) were cultured in Dulbecco's modified Eagle's medium with 10% FBS 12 , 13 , 14 , 15 . pEYFP‐canine Ku80 or pEYFP‐C1 was transiently transfected into cells using Lipofectamine 3000 (Invitrogen). Post‐transfection, cells were cultured for 2 days and then observed under an FV300 confocal laser‐scanning microscope (Olympus, Tokyo, Japan), as previously described 8 , 13 , 14 , 15 , 16 , 17 , 18 . DNA in the fixed cells was stained with 4,6‐diamino‐2‐phenylindole (DAPI) fluorescent dye, as previously described 18 .
9
Visualizing Canine Ku70 Localization
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A Madin-Darby canine kidney cell line (MDCK) (HSRRB, Osaka, Japan), a canine lung adenocarcinoma cell line (CLAC) (HSRRB), a human cervical carcinoma cell line
(HeLa) (Riken Cell Bank, Tsukuba, Japan) and a human colon cancer cell line (HCT116) (Riken Cell Bank) were cultured in Dulbecco’s modified Eagle’s medium
(DMEM) with 10% fetal bovine serum (FBS). pEYFP-canine Ku70 or pEYFP-C1 was transiently transfected into cells using Lipofectamine 3000
(Invitrogen, Carlsbad, CA, U.S.A.). Post-transfection, cells were cultured for 2 days and then monitored under an FV300 confocal laser-scanning microscope
(CLSM) (Olympus, Tokyo, Japan) as previously described [16 (link), 17 (link), 22 (link)].
(HeLa) (Riken Cell Bank, Tsukuba, Japan) and a human colon cancer cell line (HCT116) (Riken Cell Bank) were cultured in Dulbecco’s modified Eagle’s medium
(DMEM) with 10% fetal bovine serum (FBS). pEYFP-canine Ku70 or pEYFP-C1 was transiently transfected into cells using Lipofectamine 3000
(Invitrogen, Carlsbad, CA, U.S.A.). Post-transfection, cells were cultured for 2 days and then monitored under an FV300 confocal laser-scanning microscope
(CLSM) (Olympus, Tokyo, Japan) as previously described [16 (link), 17 (link), 22 (link)].
10
Quantifying Orai1-STIM1 Interactions
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The Duolink In Situ Red Starter Mouse/Rabbit kit was from Sigma (#Cat DUO92101) and used according to the manufacturer’s protocol with primary antibodies to Orai1 (mouse 1:500) and STIM1 (rabbit 1:1000). Cells (~30% confluent) were treated with thapsigargin (1 μM, 5 min) in Ca2+-free HBSS before fixation, permeabilization, and incubation with primary antibodies (16 hr, 4 °C) and the PLA reactants. Red fluorescent PLA signals were imaged using an Olympus FV300 confocal laser scanning microscope, with excitation at 561 nm, and a 60×oil-immersion objective. Quantitative analysis of the intensity and surface area of PLA spots used the ‘Analyze particle’ plugin of Fiji. Results are shown for 8–10 cells from two biological replicates of each genotype. Number of PLA spots in all genotypes and conditions were counted manually.
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