Total yeast RNA (Sigma, Gillingham, UK; Cat N° 10109223001) was prepared in 2-(N-morpholino)ethanesulfonic acid (MES) buffer (20 mM, pH 7.0) or phosphate buffer (50 mM, pH 7.5). For fluorescence microscopy, we spiked the phase-separating mixture with TDP-43 labeled with Alexa Fluor™ 488 at 1:100 ratio. We incubated the samples for 5 min (unless otherwise stated) in incubation chambers (grace biolabs, 666,106 JTR12R-A2-1.0) sealed with 1.5# coverslips coated with PEG silane. Samples were visualized by fluorescence microscopy (Nikon A1R Eclipse Ti, Tokyo, Japan) using a 488 laser and Apo 60X objective. Images were taken in approximately the middle of the incubation chamber to avoid wetting effects on the coverslips.
A1r eclipse ti
Manufactured by Nikon
Sourced in Japan
The A1R Eclipse Ti is a high-performance inverted microscope system designed for advanced biological and biomedical research applications. It features a modular design, allowing for the integration of a wide range of imaging techniques and accessories to meet the specific needs of researchers.
Automatically generated - may contain errors
Lab products found in correlation
Nucblue live readyprobes reagent, by Thermo Fisher Scientific (3 mentions)
4 6 diamidino 2 phenylindole dapi, by Yesen (2 mentions)
Total yeast rna, by Merck Group (1 mentions)
Fv1000, by Olympus (1 mentions)
Jbw301, by Merck Group (1 mentions)
Pc130, by Merck Group (1 mentions)
Labtek permanox chamber slides, by Thermo Fisher Scientific (1 mentions)
Chamber slide, by Corning (1 mentions)
Annexin 5 fitc apoptosis detection kit, by Beyotime (1 mentions)
Annexin 5 fitc, by Beyotime (1 mentions)
Nuclear extraction kit, by Solarbio (1 mentions)
Fitc labeled anti rabbit secondary antibody, by Cell Signaling Technology (1 mentions)
13 protocols using a1r eclipse ti
1
Visualizing TDP-43 Phase Separation
2
Multicolor Fluorescence Imaging of OM Channels
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Depending on substrate opacity, either an upright or inverted microscope was employed to record the fluorescence microscope images.
In particular, the upright CLSM (FV-1000, Olympus) was used for OM channels printed on a reflective substrate (e.g., silicon wafer), while the inverted CLSM (A1R Eclipse Ti, Nikon) was used for OM channels printed on a transparent substrate (e.g., cover glass). The confocal microscopes are equipped with four lasers (410, 489, 561, 638 nm) and four associated detectors. Each detector records the emission spectrum of a different wavelength range (detector 1: 425–475 nm, detector 2: 500–550 nm, detector 3: 570–620 nm, detector 4: 663–738 nm). A particular detector with the strongest signal was selected for each fluorescent dye or biomolecule. Biomolecules with distinct fluorescence colours were selected for making mixture solutions. The differentiation of biomolecules in the mixture can be achieved by using multiple detectors simultaneously. Additionally, a differential interference contrast (DIC) detector can be used to observe the shape of OM channel. More details about CLSM experiments are provided in Supplementary Table6 .
In particular, the upright CLSM (FV-1000, Olympus) was used for OM channels printed on a reflective substrate (e.g., silicon wafer), while the inverted CLSM (A1R Eclipse Ti, Nikon) was used for OM channels printed on a transparent substrate (e.g., cover glass). The confocal microscopes are equipped with four lasers (410, 489, 561, 638 nm) and four associated detectors. Each detector records the emission spectrum of a different wavelength range (detector 1: 425–475 nm, detector 2: 500–550 nm, detector 3: 570–620 nm, detector 4: 663–738 nm). A particular detector with the strongest signal was selected for each fluorescent dye or biomolecule. Biomolecules with distinct fluorescence colours were selected for making mixture solutions. The differentiation of biomolecules in the mixture can be achieved by using multiple detectors simultaneously. Additionally, a differential interference contrast (DIC) detector can be used to observe the shape of OM channel. More details about CLSM experiments are provided in Supplementary Table
3
Live Cell Imaging of PN Internalization
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Live cell imaging microscopic study was performed to visualize the internalization and retention of PN within the cells [24 ]. HeLa cells (5×104 cells/well) were cultured on glass culture dish (33 mm) and treated with PN (100 μM). Culture dish was kept in an enclosed chamber of live cell confocal microscope (Nikon, A1R eclipse-Ti) for providing an adequate cell culture conditions (37°C temperature with 5% CO2) with all other parameters with default setting. Live PN internalization, retention into nucleus from cytoplasm and apoptosis induction were visualized by NIS-Elements imaging software at 1h and 30 h post PN-treatment. In addition, we also determined the effect of PN on deformation of nucleus structure and mitochondrial membrane potential using Hoechst 33342 stain (100 μg/ml) and JC-1 probes (1 μg/ml) according to the manufacturer’s instructions [25 ]. Morphology of stained cells was visualized and imaged at 40× magnification under live cell confocal microscopy.
4
Quantifying DNA Damage and Repair
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Cells were seeded in 8-well Lab-tek Permanox Chamber slides (Nunc, Zellik, Belgium), treated for 24 hr, fixed in 4% paraformaldehyde for 15 min at room temperature, and ice-cold methanol for 5 min. Primary antibodies recognizing γH2AX (JBW301, Millipore, Overijse, Belgium) and RAD51 (PC130, Merck, Darmstadt, Germany) followed by secondary antibodies conjugated to Alexa Fluor 647 and 488 (Life Technologies) were used. Images were acquired using an A1R Eclipse Ti inverted confocal microscope (Nikon, Brussels, Belgium) and processed using Fiji software, with compound or vehicle-treated cells being processed identically. Nuclei with >5 foci were scored as positive, and at least 200 nuclei were counted per condition by two independent individuals, blinded to the genotypes.
5
Optical Density and Imaging of TDP-43 LCD
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For the optical density measurements unlabelled TDP-43_LCD (stock concentration of 40 µM in a 20mM MES buffer pH5) was mixed 1:1 with each buffer condition (50 mM buffers) and immediately transferred to a 96-well plate (Corning, half area, non-binding plate) and OD600 measurements were performed with a FluoOmega plate reader (BMG LABTECH). At least four independent repeats were performed. For the imaging experiments, purified protein stored in MES buffer was combined with tagged TDP-43_LCD. Alexa Fluor™ 488 NHS Ester was used to tag TDP-43_LCD, which was mixed with unlabelled protein at a 1:50 ratio.
The protein was added at a 1:1 ratio with buffer 1 or buffer 2 at a final concentration of 20uM. The sample left to incubate for 10 minutes before images were obtained using the Nikon A1R Eclipse Ti (Japan).
The protein was added at a 1:1 ratio with buffer 1 or buffer 2 at a final concentration of 20uM. The sample left to incubate for 10 minutes before images were obtained using the Nikon A1R Eclipse Ti (Japan).
6
Annexin V-FITC Assay for Apoptosis
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To detect apoptosis or necrosis, cells were plated in chamber slides (3 × 104 cells per well) (Corning Inc.), allowed to attach overnight, and exposed to various experimental conditions. After treatment for 72 h, the cells were washed with PBS and incubated at room temperature with Annexin V-FITC conjugates and propidium iodide, following the standard procedures for the Annexin V-FITC Apoptosis Detection Kit (Beyotime Biotechnology). After 20 min, apoptotic cells were detected with a Nikon A1R Eclipse Ti confocal microscope (Nikon Corporation, Tokyo, Japan).
7
Visualizing Arabidopsis Root Meristems
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Arabidopsis seedlings (6 d old) were stained with propidium iodide (10 μg ml−1) for 15 min before rinsing in water. Root meristems were visualized using a Nikon A1R Eclipse Ti inverted confocal microscope (Nikon, Tokyo, Japan). To determine meristem size, distance from the quiescent centre to the first elongated cortex cell was measured using Image J.
8
Visualizing Nuclear Translocation of Transcription Factors
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The nuclear localization of p65, c-Jun, and IRF3 was detected with Nikon A1R Eclipse Ti confocal microscope (Nikon Corp., Tokyo, Japan) as previously described [21] . Brie y, cells were seeded into chamber slide for 12 h, and then treated with FSI (100 and 200 μg/mL) for 1h. The cells were xed by 4% paraformaldehyde for 10 min and then permeabilized with Triton X-100 (0.25%) during 30 min at 37 °C. Cells were then blocked for 1 h with BSA (2%) and incubated with target antibodies including p65, c-Jun and IRF3 diluted in cold PBS containing 3 % BSA at 4°C for overnight. Then, the cells were incubated with 1:1,000 dilution of FITC-labeled anti-rabbit secondary antibody in dark under room temperature for 1 h. After incubating, the cells were washed for 3 times by cold PBS. 4′,6-diamidino-2-phenylindole (DAPI, YESEN, Shanghai, China) was used to stain nucleus right before performing assay.
Cytosolic and nuclear protein separation RAW264.7 cells were seeded (5×10 5 cells/well) into 60 mm-diameter culture dishes and cultured for 24 h. After treatment with FSI for 1 h and followed by LPS stimulation for 30 min, PBS was used to wash cultures, and then using nuclear extraction kit (Solarbio, Beijing, China) to separate nuclear and cytosolic protein. After that, the samples were dissolved in lysis buffer for Western blot assay.
Cytosolic and nuclear protein separation RAW264.7 cells were seeded (5×10 5 cells/well) into 60 mm-diameter culture dishes and cultured for 24 h. After treatment with FSI for 1 h and followed by LPS stimulation for 30 min, PBS was used to wash cultures, and then using nuclear extraction kit (Solarbio, Beijing, China) to separate nuclear and cytosolic protein. After that, the samples were dissolved in lysis buffer for Western blot assay.
9
Nuclear Localization of Transcription Factors
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The nuclear localization of p65, c-Jun, and IRF3 was detected with Nikon A1R Eclipse Ti confocal microscope (Nikon Corp., Tokyo, Japan) as previously described[ 24 ]. Briefly, cells were seeded into chamber slides for 12 h and then treated with flavonoids of S. involucrata (100 and 200 μg/mL) for 1 h. After LPS (1 μg/mL) stimulation for 1 h, cells were fixed by 4% paraformaldehyde for 10 min and then permeabilized with Triton X-100 (0.25%) for 30 min at 37 °C. Cells were then blocked for 1 h with 2% bovine serum albumin and incubated with target antibodies including p65, c-Jun, and IRF3 diluted in cold PBS containing 2% bovine serum albumin at 4 °C overnight. Then, the cells were incubated with FITC-labeled anti-rabbit secondary antibody (Cell Signaling Technology, catalog no. 4412; 1:500 dilution) in dark under room temperature for 1 h. After incubation, cells were washed 3 times by cold PBS. 4',6-diamidino-2-phenylindole (DAPI, YESEN, Shanghai, China) was used to stain nucleus right before performing assay.
10
Quantifying Nuclear Strain in Skeletal Muscle
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