Drosophila larvae were imaged using 3rd instar larvae 27–29 hours after the wasp infection. The larvae were washed three times in H2O and embedded on microscope slides in a drop of ice-cold glycerol. The larvae were immobilized at -20°C before imaging. The Zeiss ApoTome.2 was used for live imaging of larvae. For hemocyte imaging, the larvae were washed three times in H2O, and the hemocytes were bled into 1 x PBS 48–50 hours after the wasp infection. Uninfected controls of the same age were also used. The hemocytes were let to adhere to the glass surface of a microscope slide for 30 minutes, after which they were fixed with 3.7% paraformaldehyde for 5 minutes. The samples were washed with PBS and mounted with the Prolong Gold Anti-Fade reagent with DAPI (Molecular Probes). Hemocyte imaging was carried out with the Zeiss AxioImager.M2 microscope with Zeiss AxioCam and the Zen Blue 2011 software and with the Zeiss LSM780 in the case of the antibody-stained hemocytes. The hemocyte images were processed with ImageJ 1.49p (Rasband WS, ImageJ, U.S. National Institutes of Health, Bethesda, Maryland, USA, imagej.nih.gov/ij, 1997–2012).
Zen blue 2011
Manufactured by Zeiss
Sourced in Germany
Zen Blue 2011 is a microscope imaging and analysis software developed by Zeiss. It provides a user-friendly interface for capturing, processing, and analyzing images obtained from Zeiss microscope systems.
Automatically generated - may contain errors
Lab products found in correlation
Axio observer z1, by Zeiss (6 mentions)
Plan apochromat objective, by Zeiss (3 mentions)
Dp73 camera, by Olympus (3 mentions)
Lsm 780, by Zeiss (2 mentions)
Pluronic f 127, by Thermo Fisher Scientific (2 mentions)
Axiocam hrm3, by Zeiss (2 mentions)
Fluo 4 am, by Thermo Fisher Scientific (2 mentions)
Axio scope a1 microscope, by Zeiss (2 mentions)
Z1 axioscope microscope, by Zeiss (2 mentions)
Prolong gold antifade reagent with dapi, by Thermo Fisher Scientific (1 mentions)
Apotome 2, by Zeiss (1 mentions)
Axio imager m2 microscope, by Zeiss (1 mentions)
Axio observer z1 inverted epifluorescence microscope, by Zeiss (1 mentions)
Hoechst 33258, by Thermo Fisher Scientific (1 mentions)
Axio observer z1 fluorescent microscope, by Zeiss (1 mentions)
Indesign cc, by Adobe (1 mentions)
10 protocols using zen blue 2011
1
Drosophila Larval Imaging and Hemocyte Analysis
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Drosophila larvae were imaged using 3rd instar larvae 27–29 hours after the wasp infection. The larvae were washed three times in H2O and embedded on microscope slides in a drop of ice-cold glycerol. The larvae were immobilized at -20°C before imaging. The Zeiss ApoTome.2 was used for live imaging of larvae. For hemocyte imaging, the larvae were washed three times in H2O, and the hemocytes were bled into 1 x PBS 48–50 hours after the wasp infection. Uninfected controls of the same age were also used. The hemocytes were let to adhere to the glass surface of a microscope slide for 30 minutes, after which they were fixed with 3.7% paraformaldehyde for 5 minutes. The samples were washed with PBS and mounted with the Prolong Gold Anti-Fade reagent with DAPI (Molecular Probes). Hemocyte imaging was carried out with the Zeiss AxioImager.M2 microscope with Zeiss AxioCam and the Zen Blue 2011 software and with the Zeiss LSM780 in the case of the antibody-stained hemocytes. The hemocyte images were processed with ImageJ 1.49p (Rasband WS, ImageJ, U.S. National Institutes of Health, Bethesda, Maryland, USA, imagej.nih.gov/ij, 1997–2012).
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Drosophila larvae were imaged using 3rd instar larvae 27–29 hours after the wasp infection. The larvae were washed three times in H2O and embedded on microscope slides in a drop of ice-cold glycerol. The larvae were immobilized at -20°C before imaging. The Zeiss ApoTome.2 was used for live imaging of larvae. For hemocyte imaging, the larvae were washed three times in H2O, and the hemocytes were bled into 1 x PBS 48–50 hours after the wasp infection. Uninfected controls of the same age were also used. The hemocytes were let to adhere to the glass surface of a microscope slide for 30 minutes, after which they were fixed with 3.7% paraformaldehyde for 5 minutes. The samples were washed with PBS and mounted with the Prolong Gold Anti-Fade reagent with DAPI (Molecular Probes). Hemocyte imaging was carried out with the Zeiss AxioImager.M2 microscope with Zeiss AxioCam and the Zen Blue 2011 software and with the Zeiss LSM780 in the case of the antibody-stained hemocytes. The hemocyte images were processed with ImageJ 1.49p (Rasband WS, ImageJ, U.S. National Institutes of Health, Bethesda, Maryland, USA, imagej.nih.gov/ij, 1997–2012).
2
Wound Healing Assay with CRH Stimulation
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Cells were cultured in 24-well plates to confluence. Wounds were created with a pipette tip and washed to remove cell debris. Cells were stimulated with 10 nM or 100 nM CRH in DMEM 1% FBS. Images were acquired with a Zeiss Axio Observer Z1 Inverted Epi-fluorescence microscope, equipped with an AxioCam HRm3digital CCD camera; a Stage Controller XY STEP SMC 2009 scanning stage and an Incubator XLmulti S1 and Heating Unit XL S1 for live imaging incubation.
Images were acquired under bright field illumination every 15 min for 24 h using a 10X air objective and Zeiss Zen Blue 2011 software for image acquisition. Image analysis was performed with Fiji software, using an automated analysis macro to measure the area occupied by cells.
Images were acquired under bright field illumination every 15 min for 24 h using a 10X air objective and Zeiss Zen Blue 2011 software for image acquisition. Image analysis was performed with Fiji software, using an automated analysis macro to measure the area occupied by cells.
3
Cell Growth Kinetics Quantification
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This experiment was performed as described previously, with minor modifications [80] . Briefly, 1x10 3 cells were seeded in 96-well plates and incubated overnight to allow cell attachment. Images were then acquired under bright field illumination every 6 hours for 3 days using a 10X air objective and Zeiss Zen Blue 2011 software for image acquisition. Image analysis was performed with Fiji software, using an automated analysis macro to measure the occupied area by cells. An exponential growth model for non-lineal regression was used to calculate doubling time.
4
Automated Cell Tracking and Imaging
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The procedure was performed as described previously, with minor modifications [82] [83] [84] . A 1% noble agar solution was heated until boiling, swirled to facilitate complete dissolution, and then taken off of the heat. When the temperature cooled to 50°C, 5 μL spots were pipetted onto 96 well cell culture plates and allowed to cool for 20 min at RT under the hood. At this point, 5x10 3 cells were plated into spot-containing wells in the presence of 10% FBS cell culture media supplemented with 1 μg/ml Hoechst 33258 (Thermo Fisher Scientific) and allowed to adhere for 1 hour. Fluorescent images of the edges of each spot were taken every 20 minutes during 18 hours on an Axio Observer Z1 (Zeiss) Fluorescence Microscope using a 10X magnification air objective, equipped with CCD Axio Cam HRm3 digital Camera, and a XL multi S1 (D) incubation unit plus a XL S1 (D) temperature module to maintain cell culture conditions at 37°C and 5% CO2. Acquisition was controlled with Zen Blue 2011 (Zeiss) Software. Graph construction and statistical analysis were performed using GraphPad Prism. The number and position of cells were determined using ImageJ/Fiji -'Trackmate' plug-in [81] .
5
Visualizing Zebrafish Embryo Mutants
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Live zebrafish embryos and larvae were visualized using a Nikon MULTIZOOM AZ100 equipped with epi-fluorescence and an Andor Clara digital camera unless otherwise noted. To validate mutants with 5xERE reporter activity, larvae were treated overnight with 100 ng/mL estradiol beginning at 2–3 dpf. Following overnight treatment, larvae were washed in E3B, anesthetized with 0.04% tricaine and imaged in Petri dish containing E3B. For S1H–S1K Fig , larvae were mounted in bridged coverslips in E3B with 0.04% tricaine [74 ]. Images were captured on a Zeiss Axio Observer.Z1 fluorescent microscope equipped with an Axio HRm camera and Zen Blue 2011 software (Carl Zeiss Microscopy, Oberkochen, Germany). Adjustments, cropping and layout were performed using Photoshop CC and InDesign CC (Adobe Systems Inc., San Jose, CA).
6
Live-Imaging of Calcium Dynamics in Cells
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Cells plated in glass-bottom dishes were loaded for 30 min in darkness with 6 µM Fluo-4-AM and 0.14% Pluronic F-127 (Molecular Probes) in Ringer buffer. Images were acquired with a Axio Observer Z1 inverted epi-fluorescence microscope (ZEISS), equipped with an AxioCam HRm3 digital CCD camera, a Stage Controller XY STEP SMC 2009 scanning stage, and an Incubator XLmulti S1 (D) and Heating Unit XL S1 (D) for live-imaging incubation. Data acquisition was controlled by Zen Blue 2011 software (ZEISS), configured at a bit depth of 14 bits. Cells were imaged with a 20X air objective (Plan-Apochromat NA 0.8 M27) and illuminated using Colibri.2 470-nm LED excitation (5% power), with a 50-ms exposure acquired every 5 s and a 38HE Filter. Image analysis was performed with Fiji by measuring calcium-dependent changes in fluorescence intensity from resting levels (ΔF/F0) in 30–40 cells randomly selected in each experiment.
7
Visualizing Calcium Signaling in Cells
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For experiments using ester calcium indicators, cells plated in glass-bottom dishes were loaded for 30–60 min in darkness with 6 µM Fluo-4-AM and 0.14% Pluronic F-127 (Molecular Probes) in Ringer buffer. Images were acquired with a Axio Observer Z1 inverted Epifluorescence microscope (ZEISS), equipped with an AxioCam HRm3 digital CCD camera, a Stage Controller XY STEP SMC 2009 scanning stage, and an Incubator XLmulti S1 (D) and Heating Unit XL S1 (D) for live-imaging incubation. Data acquisition was controlled by Zen Blue 2011 software (ZEISS), configured at a bit depth of 14 bits. Cells were imaged with a 20× air objective (Plan-Apochromat NA 0.8 M27) and illuminated using Colibri.2 470-nm LED excitation (5% power), with a 50-ms exposure acquired every 5 s and a 38HE Filter set for a duration of 10 min. When indicated, cells were preincubated with different reagents. Approximately 30 s after the start of the experiment, CRH was added to a final concentration of 100 nM. Image analysis was performed with Fiji by measuring calcium-dependent changes in fluorescence intensity from resting levels (ΔF/F0) in 30–40 cells randomly selected in each experiment.
8
Fluorescence Imaging Acquisition Protocol
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Images were acquired with D1 inverted microscope (Carl Zeiss, Germany) equipped with DP73 camera (Olympus, Japan) or with Zeiss LSM 700 inverted confocal microscope (Carl Zeiss, Germany) equipped with 405nm, 488nm, 555nm and 635 solid state lasers, using a 20x Plan-Apochromat objective (NA 0.8). All images were acquired in sequential mode. For comparative analysis, all parameters during image acquisition were kept constant throughout each experiment. Images were processed with Zen blue 2011 software (Carl Zeiss, Germany), and Adobe Photoshop CS4.
9
Automated Fluorescence Image Analysis
10
Automated Fluorescence Image Analysis
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Images were acquired with A1 Axioscope microscope (Carl Zeiss) equipped with DP73 camera (Olympus) or with Z1 Axioscope microscope (Carl Zeiss), using a 20X Plan-Apochromat objective (numerical aperture 0.4). All images were acquired in sequential mode. Images were processed with Zeiss Zenblue 2011 software (Carl Zeiss) and Adobe Photoshop CS4. In house algorithm was developed and implemented in Matlab to process the images and analyze the percentage of tdTomato+/ green-fluorescence+ overlay area out of total green-fluorescence+ area (Supplementary Fig. 5c and 7 ). The algorithm consists of the following steps: 1) Noise reduction using sliding median filter. 2) Creating a binary mask image per channel using a detection threshold defined as the overall image median value plus bias (defined as 10% of the dynamic range). All pixels with values above the threshold are “detected” 3) Overlaying tdTomato+ and green-fluorescence+ mask images and calculating the percentage of tdTomato+ & green-fluorescence+ pixels out of all green-fluorescence+ pixels.
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