Double or single fluorescent immunohistochemistry was performed to examine the colocalization of CD133, 8-nitroguanine, and 8-oxodG as described previously [25 (link)]. Paraffin sections were incubated with the primary antibodies [rabbit polyclonal anti-CD133 antibody (1 : 500, Abcam, Cambridge, UK), rabbit polyclonal anti-8-nitroguanine antibody (1 μg/mL) produced by our group [25 (link), 26 (link)], and mouse monoclonal anti-8-oxodG antibody (1 : 200, Japan Institute for the Control of Aging, Fukuroi, Japan)] overnight at room temperature. The sections were next incubated with fluorescent secondary antibodies (Alexa 488-labeled goat anti-mouse IgG and/or Alexa 594-labeled goat anti-rabbit IgG antibodies, 1 : 400 each, Molecular Probes Inc., Eugene, Oregon, USA) for 3 h at room temperature. Finally, the nuclei were stained by 4′-6-diamidino-2-phenylindole (DAPI) and the sections were examined with a fluorescence microscope (LX70, Olympus, Tokyo, Japan) or a laser scanning confocal microscope (Fluoview FV1000-D, Olympus) [10 (link)].
Fluoview fv1000d
Manufactured by Olympus
Sourced in Japan
The Fluoview FV1000D is a confocal laser scanning microscope system designed for high-resolution imaging of fluorescent samples. It features a modular design, allowing for customization to meet specific research requirements.
Automatically generated - may contain errors
Lab products found in correlation
Bz 9000, by Keyence (3 mentions)
Hoechst 33342, by Merck Group (3 mentions)
Metamorph software, by Molecular Devices (2 mentions)
Alexa 488 labeled goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 594 goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Anti brdu, by Roche (1 mentions)
Vectashield mounting medium, by Vector Laboratories (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Vectashield vibrance antifade mounting medium, by Vector Laboratories (1 mentions)
Fluorescein conjugated secondary antibody, by Merck Group (1 mentions)
Dapi 4 6 diamidino 2 phenylindole, by Merck Group (1 mentions)
Alexa fluor 488 labeled goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Mouse anti β catenin antibody, by BD (1 mentions)
Guinea pig anti insulin antibody, by Agilent Technologies (1 mentions)
Blocking one, by Nacalai Tesque (1 mentions)
Alexa fluor 488 donkey anti goat igg, by Thermo Fisher Scientific (1 mentions)
Bz 2 analyzer software, by Keyence (1 mentions)
In cell analyzer 6000, by GE Healthcare (1 mentions)
Tissue tek oct compound, by Sakura Finetek (1 mentions)
22 protocols using fluoview fv1000d
1
Colocalization of CD133, 8-Nitroguanine, and 8-Oxodg
2
Identification of Adult Neurogenesis by DCX and BrdU Labeling
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Doublecortin (DCX) is one of the suitable markers for adult neurogenesis [50] (link). For double labeling with BrdU and DCX, sections were cut at a thickness of 40 µm. After pretreatment with 2 N HCl, the sections were incubated overnight at 4°C in anti-BrdU (1∶100; Roche Diagnostics) and rabbit anti-DCX antibodies (1∶300; Cell Signaling Technology, Danvers, MA) and then incubated for 1 h at room temperature in Alexa Fluor 488 goat anti-mouse IgG and Alexa Fluor 594 goat anti-rabbit IgG (1∶2,000; Invitrogen, Carlsbad, CA). The sections were then mounted with VECTASHIELD Mounting Medium (Vector Laboratories Inc., Burlingame, CA). Individual double-labeled cells in the SVZ were visualized using a confocal fluorescence microscope (Fluoview FV1000-D; Olympus, Tokyo, Japan).
3
Visualizing Autophagy in Phafin2-Modulated J774.1 Cells
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Phafin2 siRNA and/or retroviral human Phafin2-introduced J774.1 cells were treated with Premo™ Autophagy Sensors (LC3B-GFP) for 16 hours [30] (link). The cells were then incubated with Alexa Fluor 594-killed E.coli (E-23370, Escherichia coli Bioparticles®, Molecular Probes) at 10 µg/ml for 1 hour at 37°C, washed 3 times with pre-warmed PBS and incubated in HBSS for 4 hr to induce autophagy. Cells were fixed with 3.7% formaldehyde immediately after 4 hr incubation in HBSS. The cells were subsequently stained with DAPI (4′, 6-diamidino-2-phenylindole, blue, Sigma) and observed using confocal microscopy (FLUOVIEW FV1000-D, Olympus). The white scale bar represents 10 µm. LC3 puncta per cells were counted in 20 cells and shown as a bar graph. Statistical analysis was conducted via Student's t-test.
4
Immunofluorescence Staining of Brain Sections
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The sections were incubated in methanol for 5 min at −20 ℃, following which they were incubated in 10% NGS. Thereafter, the samples were incubated with Alexa Fluor 594-labeled goat anti-human IgG (1:200) antibody for 60 min in the dark. Finally, they were stained with DAPI (1:40,000; Molecular Probes, Eugene, OR) for 5 min and enclosed using Vectashield Vibrance Antifade mounting medium (H-1700; Vector Laboratories). The sections were observed in the aid of florescence microscope Axio Imager M1.
The sections were incubated in ice-cold methanol for 15 min, following which non-specific reaction was blocked with 10% NGS. Thereafter, the samples were incubated rat anti-MBP (1:200) overnight. They were then incubated with Alexa Fluor 488-labeled goat anti-rat IgG antibody (1:200), and also with Alexa Fluor 594-labeled goat anti-human IgG (1:200) antibody to detect IVIg. The sections were stained with DAPI and enclosed as described above. The sections were observed in the aid of confocal microscope FLUOVIEW FV1000-D (Olympus Corporation, Tokyo, Japan). All antibodies used are listed in Table1 .
The sections were incubated in ice-cold methanol for 15 min, following which non-specific reaction was blocked with 10% NGS. Thereafter, the samples were incubated rat anti-MBP (1:200) overnight. They were then incubated with Alexa Fluor 488-labeled goat anti-rat IgG antibody (1:200), and also with Alexa Fluor 594-labeled goat anti-human IgG (1:200) antibody to detect IVIg. The sections were stained with DAPI and enclosed as described above. The sections were observed in the aid of confocal microscope FLUOVIEW FV1000-D (Olympus Corporation, Tokyo, Japan). All antibodies used are listed in Table
5
Immunostaining of MIN6c4 cells
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MIN6c4 cells were fixed with 4% paraformaldehyde for 10 min and then permeabilized in PBS containing 0.2% Triton X-100 for 10 min at room temperature. The cells were blocked using Blocking One (Nacalai Tesque, Kyoto, Japan) for 60 min at room temperature followed by incubation with a primary antibody at 4°C overnight and then with a fluorescein-conjugated secondary antibody (1:400) and DAPI (4’, 6-diamidino-2-phenylindole) (Sigma-Aldrich, Saint Louis, MO, USA) for 60 min at room temperature. The stained cells were examined by confocal laser scanning microscopy using a FLUOVIEW FV1000D (Olympus, Tokyo, Japan). The following primary antibodies were used in this analysis: purified rabbit anti-TMEM59L antibody (1:400), guinea pig anti-insulin antibody (1:500, DAKO, Glostrup, Denmark), mouse anti-GM130 antibody (1:500, BD Transduction Laboratories, San Jose, CA, USA), and mouse anti-β-catenin antibody (1:1,000, BD Transduction Laboratories). The following secondary antibodies were used in this analysis: Alexa Fluor 488-labeled goat anti-rabbit IgG, Alexa Fluor 594-labeled goat anti-guinea pig IgG, and Alexa Fluor 647-labeled goat anti-mouse IgG1 (Life Technologies, Grand Island, NY, USA).
6
Live Adipocyte Imaging via Confocal Microscopy
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For living adipocytes imaging, time-lapse microscopic technique was introduced by using the confocal laser scanning microscopy FLUOVIEW FV1000-D (Olympus Corporation, Tokyo, Japan). 3T3-L1 adipocytes were reseeded on 2 chamber dishes at 24 hrs after adenovirus infection and on day 7 after differentiation subjected to time-lapse imaging from 24 to 44 hrs after reseeding. A sealed dish of CAR-3T3-L1 adipocytes was placed in an acrylic resin box in which temperature was maintained at 37°C on the sample stage, and time-lapse images were captured every 6 min. The series of captured images were converted into a movie using MetaMorph® software (Molecular Devices, Sunnyvale, California).
7
Immunofluorescence Analysis of FSTL1, IL-1β, and TNF-α
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Formalin-fixed and paraffin-embedded (FFPE) sections were deparaffinized and rehydrated before antigen retrieval and blocking. Sections were co-incubated with the antibodies for FSTL1 (1:200, Abcam, Cambridge, MA, UK) and IL-1β (1:200, Abcam) or FSTL1 and TNF-α (1:200, Abcam). As a secondary antibody-only control, PBS was used instead of primary antibodies. After overnight incubation at 4°C, slides were washed with PBS and incubated with secondary antibodies (1:400, Alexa Fluor 488 donkey anti-goat IgG, Molecular Probes, or Rhodamine 568-labeled anti-rabbit IgG, Chemicon International, Temecula, CA, USA) for 1 h at room temperature. Finally, nuclei were counterstained with 4′-6-diamidino-2-phenylindole (DAPI) and sections were examined under a confocal laser scanning microscope (Fluoview FV1000-D, Olympus, Tokyo, Japan). H & E staining for histology analysis was performed as described [35 (link)]. Immunohistochemistry staining was done as we described before [36 (link)].
8
Quantifying Graft Integration and Axon Tracing
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Images were visualized using a fluorescence microscope (BZ-9000; Keyence, Osaka, Japan), In Cell Analyzer 6000 (GE Healthcare) and confocal laser microscope (Fluoview FV1000D; Olympus). To measure cell counts, immunopositive cells were manually counted for at least three independent samples to calculate the age of positive cells for each marker. The number of immunopositive cells in the graft was quantified in every 6 sections and corrected. To measure the graft size, low magnified GFP images were imported into the BZ-II Analyzer software (Keyence), and the graft areas were quantified every six sections. The estimated graft volumes were calculated based on the thickness of the brain slices. The number of axons derived from a graft was counted in the coronal section at the internal capsule and the cerebral peduncle. The site of interest in each animal was labeled using an anti-GFP antibody, and the mean number of axons was recorded.
9
Immunohistochemistry of Nox1 KO Mouse Testes
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For immunohistochemistry of Nox1 KO mouse testes, the samples were fixed in 4% paraformaldehyde for 2 h. They were then embedded in Tissue-Tek OCT Compound (Sakura Finetek) for cryosectioning. Immunostaining of cryosections was performed by treating samples with 0.1% Triton-X in PBS. For staining GS cells, single-cell suspensions were concentrated on glass slides by centrifugation using a Cytospin 4 unit (Thermo Electron Corp.). After immersing the slides in blocking buffer (0.1% Tween 20, 1% bovine serum albumin, and 10% donkey serum in PBS) for >1 h, the samples were incubated with antibodies against the indicated antigens. Secondary antibodies were incubated for 1 h at room temperature. The samples were counterstained with Hoechst 33342 (Sigma). The images were collected using confocal microscope (Fluoview FV1000D; Olympus). Images were analyzed by Photoshop software (Adobe Systems). Quantification of BCL6B levels was performed using MetaMorph software (Molecular Devices). All antibodies are listed in Table S9.
Table S9
10
Cytokine-Induced β-Cell Apoptosis
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Islets were pre-cultured alone or with 5 or 10 nmol/L SDF-1 for 48 h and then cultured with mixed cytokines (50 U/mL IL-1β, 1000 U/mL IFN-γ, and 1000 U/mL TNF-α) for the final 20 h of incubation. Islets cultured without cytokines served as controls. TdT-mediated dUTP nick-end labeling (TUNEL) analysis was used to evaluate β-cell apoptosis as described previously [30 (link)]. Islets were assayed on poly-l -lysine slides using an ApopTag in situ detection kit (Millipore, Burlington, MA, USA). Images were obtained using a confocal laser-scanning microscope (FluoView FV1000-D, Olympus, Tokyo, Japan).
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