For confocal microscopy, cell pellets were prepared as in the propidium iodide assays with 500 μL of the culture transferred to a 1.5 mL microcentrifuge tube containing Compound 2 to give a final concentration of 2 μM. Samples were incubated at 30°C for 30 min and cells pelleted by centrifugation at 17,000 g for 4 min, then resuspended in 200 μL of Baclight solution containing 10 μM SYTO9 and 60 μM PI. A 10 μL sample was applied to a 1 cm x 1 cm 1.5% agarose (Bioline) pad on a microscope slide with a cover slip placed on top. The slide was imaged using a 63× lens on a confocal microscope (Zeiss 800 Airyscan) with Compound 2 being imaged with the airyscan function using a 405 nm laser and an emission filter of 450–550 nm, SYTO 9 using a 488 nm laser and 550–580 nm filter and PI using a 488 nm laser and an emission filter of 600–650 nm. Samples were irradiated using the 405 nm laser at 30% power for 1 min (total energy: 90 mJ/cm2) on the microscope. A time lapse video was assembled from images taken every 6 s for 10 min.
Airyscan 800
Manufactured by Zeiss
The Airyscan 800 is a high-resolution confocal imaging system developed by Zeiss. It utilizes an Airyscan detector to provide enhanced resolution and sensitivity compared to traditional confocal microscopy. The core function of the Airyscan 800 is to enable high-quality imaging of biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Zen blue software, by Zeiss (4 mentions)
Las x life science software, by Leica (1 mentions)
Silencer sirna labelling kit, by Thermo Fisher Scientific (1 mentions)
Chamber slide, by Merck Group (1 mentions)
Alexa fluor 568, by Thermo Fisher Scientific (1 mentions)
Vectashield hardset antifade mounting medium, by Vector Laboratories (1 mentions)
Af676, by R&D Systems (1 mentions)
Nb300 317, by Novus Biologicals (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 594, by Thermo Fisher Scientific (1 mentions)
Thunder imager model organism, by Leica (1 mentions)
8 protocols using airyscan 800
1
Confocal Imaging of Compound 2 Effects
2
Polarized Laser Actuation of Planar Samples
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The planar samples were imaged and actuated under a confocal microscope (Zeiss 800 Airyscan, Objective: Plan-Achromat 63x oil, NA = 1.4) using the 561 nm laser line for continuous scanning illumination (writing mode, default: linear horizontal polarization) and the 640 nm line for transmission or scattered laser beam imaging (reading mode) of the sample. For actuation with circular polarization, a λ/4 plate was inserted in the optical path via the beam-blocker slit and circular polarization of the laser beam was verified in polarized optical microscopy (POM) mode. The waveplate was removed for all image acquisitions. Irradiation powers for planar substrates were defined as laser power divided by the total rectangular scanning/illumination area (33.8 × 33.8 – 101.4 × 101.4 µm2) and in writing mode the scanning speed was set to its maximal value (pixel dwell time 0.59–0.91 µs, frame time 233–360 ms). Time-delay between the end of a writing illumination step and onset of the next writing interval was approximately 45 s.
3
Quantifying RAP2.12-GFP Fluorescence in Botrytis-Infected Cells
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RAP2.12‐GFP fluorescence was imaged post‐infection with B. cinerea using a Zeiss Airyscan 800 confocal microscope (GFP, green fluorescent protein). Zen blue software (Carl Zeiss, Oberkochen, Germany) was used to quantify RAP2.12‐GFP fluorescence intensity. Five individual biological replicates were used for each treatment. Each data point represents the average fluorescence intensity at the nuclei or plasma membrane of all analyzed cells in one biological replicate. Boxplot limits represent the 25th and 75th percentiles of each dataset. The whiskers extend to the lowest and highest data point within the 1.5 × interquartile range of the 25th and 75th quartile. The central line represents the median.
4
GFP-PCO Imaging in Nicotiana benthamiana
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For GFP‐PCO imaging, the abaxial side of leaves from transiently transfected Nicotiana benthamiana (4 weeks old) were analysed with a Leica DM6000B/SP8 confocal microscope (Leica Microsystems) using 488‐nm laser light (20% laser transmissivity), photomultiplier tube detection, and emission light was collected between 491 and 551 nm. Images were analysed and exported using the LAS X life science software (www.leica-microsystems.com ), with an unchanged lookup table settings for each channel. Imaging of the ratiometric mClover3‐Ubi‐RAP2.32–70‐mRuby reporter was performed using a Zeiss airyscan 800. Fiji was used to quantify mClover3 and mRuby mean fluorescence intensity in protoplast nuclei.
5
Visualizing miRNA Uptake in Roots
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Synthetic miRNA was labelled with Cy3 using the Silencer siRNA labelling kit (Thermo Fisher Scientific) in accordance with the manufacturer’s instructions. Five-day-old Col-0 seedlings grown in vertical plates (MS half strength, 0.5% sucrose and 0.7% plant agar) were treated by applying 9.6 µl of the labelled miRNA (20 µM) along the roots. After 2 h incubation, confocal microscopy was performed. Samples were washed two or three times with distilled water to prevent labelled miRNA from attaching to the outer root surface. To verify that the fluorescence was emitted by the miRNA and not the free dye, a blank labelling reaction was performed (using water instead of miRNA), and plants were treated with this blank reaction. Treated roots were imaged using a Zeiss Airyscan 800 laser scanning confocal microscope. Cy3 miRNA was excited using a 561 nm laser and fluorescence was detected at 564–620 nm. pSUC2:YFP and pS18:YFP fluorescence was detected using a 488 nm laser and 492–550 nm emission wavelength. Sequential scanning was used to eliminate bleed-through.
6
Immunofluorescence Staining of Cellular Proteins
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Cells grown on chamber slides (Millipore) were fixed in 4% paraformaldehyde for 10 minutes at room temperature, and rinsed three times in PBS between each of the following steps. Cells were permeabilized with 0.2% Triton X-100 in PBS for 10 minutes at room temperature (this step was omitted for surface xCT staining) and blocked with 5% BSA in PBS for 1 hour. Then, cells were incubated at room temperature for 1 hour with primary antibodies against IL1RAP (1:200, AF676, R&D Systems) and/or CD98 (1:200, H00006520-D01P, Novus Biologicals), and/or HA-tag (1:500, #3724, Cell Signaling Technology), and/or xCT (1:500, NB300-317, Novus Biologicals). Cells were subsequently incubated at room temperature for 1 hour with respective IgG (H + L) secondary antibodies conjugated with Alexa Fluor 488, Alexa Fluor 568, and/or Alexa Fluor 594 (Molecular Probes, all 1:300). All antibodies were diluted in 5% BSA in PBS. Then, cells were rinsed with PBS three times, and nuclei were counterstained with DAPI within the VECTASHIELD Hardset Antifade Mounting Medium (Vector Laboratories). The images were taken on an LSM Airyscan 800 confocal microscope using a 63× oil immersion objective and the Zen Blue software (Zeiss).
7
Quantifying Hypoxic Responses via Imaging
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Seven-days old seedlings were kept for 16 h at either 1%, 2.5%, or 21% v/v oxygen concentration and then used for GFP imaging. Imaging was performed with a Leica THUNDER imager model organism using bandpass filters for GFP (excitation: 470/40 nm, emission: 525/50 nm) and RFP (excitation: 546/10 nm, emission: 605/70 nm). Confocal laser scanning microscopy was performed using a Zeiss airyscan 800. Fiji was used to quantify UnaG and mCherry fluorescence intensity [38 (link)]. Each data point represents the average mCherry/UnaG ratio at the nuclei and cytosol.
8
Immunofluorescence Quantification of Stem Cell Markers
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Cells were washed with PBS and fixed in 4% Formaldehyde (Thermofisher, 28906) at 4℃ for 15 minutes. Afterwards cells were washed 3 times with PBS and permeabilized with 0.1% Triton X-100 for 15 minutes. Cells were blocked in 0.1 % BSA in 0.05% Triton-X-100 in PBS solution for 1 hour, before incubation with primary antibodies at 4℃ overnight. The source of the antibodies and their dilution in 0.1 % BSA in 0.05% Triton-X-100 in PBS can be found in the table below. The next day, cells were washed 3 times with PBS. Secondary antibodies were added for 2 hours, before the cells were washed again 3 times with PBS. After cells were dried for 5 minutes Duolink® In Situ Mounting Medium with DAPI (Sigma, DUO82040) was added. Slides were imaged using a Zeiss AiryScan800 laser scanning confocal microscope. Lasers and filters were chosen to illuminate at fluorophore excitation maximum. Images were processed using the software ImageJ. Illumination and gain settings were kept constant for all experiments. For the cell analysis, the software CellProfiler was used. To quantify the differentiation markers (SOX2, OCT4, NESTIN and MAP2) mean fluorescence intensity in each image, corrected for the number of cells, were measured. The mean intensity of GFP expression was measured for each cell.
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