Five representative BnaAMT proteins were cloned to generate constructs for subcellular localization analysis in Arabidopsis protoplasts. The ORF of each BnaAMT gene was amplified for insertion into the pMDC43 vector with HindIII/KpnI and XbaI to generate BnaAMT-GFP fusion proteins driven by the CaMV 35S promoter. The gene-specific primers are listed in Table S2 . The vectors were respectively transformed into Arabidopsis protoplasts, which were isolated from 4-week-old leaves according to previous work [31 (link)]. After transfection and incubation in a plate under weak light for 12–16 h, fluorescent cells were imaged using a laser scanning confocal microscope (OLYMPUS FV10-ASW, Olympus, Tokyo, Japan).
Fv10 asw
Manufactured by Olympus
Sourced in Japan, United States
The FV10-ASW is a software application for Olympus confocal microscopes. It provides advanced image acquisition, processing, and analysis capabilities for researchers and scientists.
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Lab products found in correlation
Fv1000, by Olympus (7 mentions)
Fv1200, by Olympus (4 mentions)
Fluoview fv1000 confocal microscope, by Olympus (3 mentions)
Fv1000 confocal laser scanning microscope, by Olympus (2 mentions)
Fluoview fv1000, by Olympus (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions)
Fish kit, by Boster Bio (2 mentions)
Lsm 510 meta, by Zeiss (2 mentions)
Eclipse 80i microscope, by Nikon (2 mentions)
Ab190377, by Abcam (1 mentions)
4 6 diamidino 2 phenylindole dihydrochloride, by Merck Group (1 mentions)
Fds1010, by Thorlabs (1 mentions)
Hf2li, by Zurich Instruments (1 mentions)
Uplansapo, by Olympus (1 mentions)
Syto9, by Thermo Fisher Scientific (1 mentions)
Sucrose, by Merck Group (1 mentions)
Anti nalp3 antibody, by Abcam (1 mentions)
U rfl t, by Olympus (1 mentions)
Mpe fv1000, by Olympus (1 mentions)
Elyra s 1, by Zeiss (1 mentions)
99 protocols using fv10 asw
1
Subcellular Localization of BnaAMT Proteins
2
Confocal Microscopy Evaluation of HMGB1 Translocation in SAH
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Confocal microscopy was used to observe HMGB1 translocation after SAH. Brain sections were prepared as described in the immunohistochemistry. Following rehydration, sections were incubated with 0.2% Triton X-100 in PBS for 5 min at room temperature and washed with PBS 3 times for 5 min. Sections were placed in 10 mmol/l citrate buffer (pH 6.0) and heated in the microwave oven at 95°C for 30 min. Sections were cooled at room temperature for 20 min and rinsed in PBS. Nonspecific protein binding was blocked by incubation in 5% bovine serum for 40 min. Sections were incubated with primary rabbit anti-HMGB1 (1:200; ab190377; Abcam) overnight at 4°C, followed by washing with PBS 3 times for 5 min. Sections were incubated with goat anti-rabbit secondary antibody (1:100) conjugated with fluorescein isothiocyanate and tetramethylrhodamine (cat. no. ZF-0311; Beijing Zhongshan Golden Bridge Biotechnology, Beijing, China) at 37°C for 1 h. The cell nucleus was stained with 4,6′-diamidino-2-phenylindole, dihydrochloride (1:1,000; Sigma-Aldrich; Merck KGaA). Immunofluorescence was detected using a laser scanning confocal microscope (Olympus FV10-ASW; Olympus Corp., Tokyo, Japan).
3
Home-built Stimulated Raman Scattering Microscopy
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The home-built SRS system used a pump laser integrated OPO (picoEmerald, APE, Germany). It provided 2 spatially and temporally overlapped pulse trains, with the synchronized repetition rate of 80 MHz. One beam, fixed at 1,064 nm, was used as the Stokes light. The other beam, tunable from 780 to 990 nm, served as the pump light. The intensity of the Stokes beam was modulated at 20.2 MHz by a resonant electro-optical modulator (EOM). The overlapped lights were directed into an inverted multiphoton scanning microscope (FV1000, Olympus, Japan). The collinear laser beams were focused into the sample by a 20× objective (UPlanSAPO, NA 0.75, Olympus, Japan). Transmitted light was collected by a condenser (NA 0.9, Olympus, Japan). After filtering out the Stokes beam, the pump beam was directed onto a large area photo diode (FDS1010, Thorlabs, USA). The voltage from photo diode was sent into lock-in amplifier (HF2LI, Zurich Instruments, Switzerland) to extract the SRS signal. Image was reconstructed through software provided by manufacture (FV10ASW, Olympus, Japan).
4
Confocal Microscopy Imaging and Colocalization Analysis
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The confocal microscopy imaging was performed on Olympus Fluoview FV1000 confocal microscope equipped with FV10-ASW (version 2.0 software), using the 63X oil objective (numerical aperture 1.4). Optical single sections were acquired with a scanning mode format of 1024x1024 pixels, with a pixel size of 0.21 μm sampling, speed of 40 μs/pixel and 12 bits/pixel images. Acquisition of automated-sequential collection of multi-channel images was performed in order to reduce spectral crosstalk between channels. Lasers' power, beam splitters, filter settings, pinhole diameters (equivalent to 1 Airy unit) and scan mode were the same for all examined samples of each staining. Colocalization analysis was performed using ImageJ software downloaded from http://rsb.info.nih.gov/ij (JACoP plugin).
Tables of images were processed using Adobe Photoshop CS6 software (Adobe Systems Inc).
Tables of images were processed using Adobe Photoshop CS6 software (Adobe Systems Inc).
5
Quantifying Bacterial Biofilm Viability
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The bacterial biofilms grown on the randomly selected dentin slabs were viewed using CLSM to characterize the viability of the formed biofilms. Briefly, a diluted mixture of equal volumes of SYTO 9 and propidium iodide dyes (Molecular Probes, Eugene, OR) was prepared according to the manufacturer's instructions. The biofilm on each dentin slab was then stained with 200 µl of the mixture, stored in the dark for 15 minutes, and viewed under CLSM (FV1000; Olympus Corp, Center Valley, PA). Dedicated software (FV10-ASW, Olympus Corp) was used to obtain images from 3 randomly scanned biofilm areas on each dentin sample. Imaris Software (version 7.7; Bitplane, South Windsor, CT) was used for live/dead bacterial quantifications and 3-dimensional biofilms construction.
6
Fluorescence Imaging of HT-22 Cells
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HT-22 cells were cultured on coverglass (Lab-TekH II Chambered Coverglass, Nale Nunc, Naperville, USA). After being treated with high glucose, SAMe, NaHS or resveratrol as above mentioned, HT-22 cells were incubated with 10 μM NIR-NP for 30 min and Na 2 S (50 μM and 100 μM) for following 20 min. The Olympus FV1000 confocal laser scanning microscope was used to obtain confocal uorescence images. Images and data were analyzed using Olympus software (FV10-ASW).
7
Immunohistochemical Analysis of Mouse Brain Tissue
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Mice were administered ketamine and xylazine (150 mg/kg and 10 mg/kg, respectively) and perfused with 0.1 M PB followed by ice-cold 4% paraformaldehyde (PFA). Brains were postfixed in 4% PFA overnight at 4 °C. Brain tissues were cryoprotected in 10, 20, 30% (w/v) sucrose (Sigma) before frozen in optimal cutting temperature compound. Next, Brain slices (30 μm in thickness) were collected and processed as floating slices. Primary antibodies (see Additional file 1 : Table S1 for details), the immunoreactivity of which was determined before use, were diluted in blocking solution (0.1% [v/v] Triton X-100 and 10% fetal calf serum in 0.01 M PBS) and applied to slices overnight at 4 °C. Negative controls were performed by replacing the primary antibodies with normal rabbit serum. On the next day, brain slides were washed 3 times with PBS and incubated for 2 h with secondary antibodies (see Additional file 2 : Table S2 for details). Finally, brain slices were mounted on slides. Mounted slides were imaged using an inverted laser confocal microscope (FV1000; Olympus). Cells were counted using FV10-ASW (Olympus) and Image J (NIH, http://imagej.nih.gov/ij ).
8
Immunofluorescence Analysis of NLRP3 Inflammasome in Mice
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Representative coronal brain sections of mice were obtained at 24 h after Sham operation or tMCAO (n = 3 for each group). Mice were perfused with 0.9% NaCl quickly followed by 4% paraformaldehyde in PBS. Fixed frozen cerebral sections (30 μm thick) were blocked with 10% goat serum for 1 h and then incubated overnight at 4°C with primary antibodies from Abcam including anti-NALP3 antibody (1 : 100); anti-ASC antibody (1 : 100); anti-caspase-1 antibody (1 : 100); and anti-IL-1β antibody (1 : 100), together with anti-NeuN (1 : 200, Sternberger Monoclonal Incorporation, Lutherville, MD, USA). After 3 washes with PBS, they were incubated with secondary antibodies (1 : 100, Zhongshan Biology Technology Company, China) at 37°C for 2 h. Immunofluorescence was visualized using a Laser Scanning Confocal Microscope (Olympus FV10-ASW, Japan).
9
Confocal and Nomarski Microscopy of C. elegans Gonad
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Fluorescence images were acquired using an inverted microscope (IX81; Olympus) equipped with a laser scanning confocal imaging system (FluoView FV1000; Olympus). Nomarski images were collected concurrently or alone using the same microscope using Nomarski optics. A multiline argon laser and image analysis system (FV10-ASW; Olympus) were also used for image acquisition. To analyze gonadal arm extension, lag-2p::GFP fluorescence was observed using a stereo microscope (SZX7; Olympus) equipped with a U-RFL-T 100W mercury lamp (U-RFL-T; Olympus).
10
Fluorescent Probe Localization Assay
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Cells were washed with PBS and then fixed with 4% paraformaldehyde in phosphate buffer (pH 7.4) for 20 mins at room temperature. After rinsing with PBST and blocking with 3% bovine serum albumin (BSA) in PBST for 1 hr, cells were treated with 10 μM fluorescent probe with Gefitinib quinazoline for another 3 hrs. When staining for the nuclei, cells were rinsed with PBST and treated with DAPI for 15 mins. After rinsing by PBS, cells were visualized by the laser scanning confocal microscope FV10-ASW (Ver 2.1, Olympus Corp, MPE FV1000) for the co-localized detection.
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