For spheroid formation, 104 cells in 100 µl growth medium were seeded in spheroid microplates (96-well, Corning Inc., New York, NY, USA) for 15 days. Spheroid growth was monitored daily with a Zeiss Axio Vert.A1 microscope (Carl Zeiss Microscopy Deutschland GmbH, Oberkochen, Germany) and spheroid diameters were measured (Zen blue software, V2.6, Carl Zeiss Microscopy Deutschland GmbH). The number of cells per spheroid was assessed after 7 days by disaggregating 3 spheroids per sample (StemPro™ Accutase™, Thermo FisherTM) and counting the cells manually with a Bürker chamber. Spheroid cell viability was examined using the CellTiter-Glo® 3D Cell Viability Assay (Promega, Madison, WI, USA) according to the manufacturer’s protocol. For the spheroid outgrowth assay, 4-day old spheroids were embedded in Matrigel® (Corning®) covered with growth medium and the growth was monitored daily for 14 (HCT-116), 18 (HT-29) and 19 (U-2 OS) days using a Zeiss Axio Vert.A1 microscope (Carl Zeiss Microscopy Deutschland GmbH). The diameter of the spheroids was measured using the Zen blue software (version 2.6, Carl Zeiss Microscopy Deutschland GmbH).
Zen blue software
Manufactured by Zeiss
Sourced in Germany, United States, Japan, Canada, Switzerland, Netherlands, France
Zen Blue software is an imaging and analysis platform developed by Zeiss. It provides tools for controlling and operating Zeiss microscopy hardware, as well as for image acquisition, processing, and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Axio observer z1, by Zeiss (117 mentions)
Lsm 880, by Zeiss (60 mentions)
Lsm 880 confocal microscope, by Zeiss (56 mentions)
Lsm800 confocal microscope, by Zeiss (50 mentions)
Lsm 780 confocal microscope, by Zeiss (42 mentions)
Lsm 800, by Zeiss (40 mentions)
Lsm 700, by Zeiss (36 mentions)
Lsm 780, by Zeiss (32 mentions)
Zen black software, by Zeiss (30 mentions)
Lsm 710, by Zeiss (28 mentions)
Lsm 710 confocal microscope, by Zeiss (24 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (24 mentions)
Axio scan z1, by Zeiss (21 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (18 mentions)
Axio observer, by Zeiss (16 mentions)
Hoechst 33342, by Thermo Fisher Scientific (16 mentions)
Paraformaldehyde (pfa), by Merck Group (14 mentions)
Plan apochromat, by Zeiss (14 mentions)
Lsm 980, by Zeiss (14 mentions)
Axio scan z1 slide scanner, by Zeiss (13 mentions)
487 protocols using zen blue software
1
Spheroid Formation and Outgrowth Assay
2
Measurement of Tissue Volumes and White Matter Thickness after Injury
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Sixteen days after the injury (at P21) brains were perfused, frozen and entirely cut into series of 20 μm coronal sections spaced at 500μm disposed in series. On a cresyl violet-stained series, the ipsilateral volume of the total surviving tissue, the cortex and the lateral ventricle were measured using the Zen Blue software (Zeiss). The volumes were then expressed as a percentage of the total brain volume.
WM thickness were measured on 3 consecutive cresyl violet-stained sections starting from the first on which the genu of the corpus callosum appeared (approx. 0.6–0.8 mm anterior to the Bregma according to the “atlas of the rat brain in stereotaxic coordinates at P21” of Khazipov et al.,http://www.ialdevelopmentalneurobiology.com/images/atlases/Atlas-p21.pdf 81 (link)). The thickness of the ipsilateral corpus callosum (on the midline) and the subcortical WM at the level of the cingulum (1.4 mm apart from the midline) and at the beginning of the external capsule (2 mm apart from the midline) were measured parallel to the midline with the Zen Blue software (Zeiss). Values are expressed as a mean of the three measures.
WM thickness were measured on 3 consecutive cresyl violet-stained sections starting from the first on which the genu of the corpus callosum appeared (approx. 0.6–0.8 mm anterior to the Bregma according to the “atlas of the rat brain in stereotaxic coordinates at P21” of Khazipov et al.,
3
Live Cell Imaging of Mitotic Dynamics
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For live cell imaging, cells were grown in 12-well glass bottom plates (MatTek, Ashland, MA, USA). Time lapse live cell imaging was performed on a Zeiss Cell Observer inverted wide field microscope, with 20 × 0.8 NA air objective, at 37 °C, 10% CO2 and atmospheric oxygen. Image capture commenced 24 hours post transfection, with images taken every six minutes for a duration of sixty hours using an Axiocam 506 monochromatic camera (Zeiss, Oberkochen, Germany) and Zen Blue software (v1.0, Zeiss). A Zeiss HXP 120C mercury short-arc lamp and compatible filter cubes were used to obtain fluorescent images and differential interference contrast (DIC) microscopy to capture brightfield images. To achieve optimal image resolution without excessive illumination, a binning factor was applied prior to imaging and the ambient conditions were maintained to minimize variations in optical resolution and illumination. The acquired videos were analyzed using the Zen Blue software (v1.0, Zeiss). For all videos, mitotic duration and outcomes were scored by eye and calculated from nuclear envelope breakdown until cytokinesis. FUCCI videos were scored by eye, for G1 (red) and S/G2/M (green) and EBFP2 (blue).
4
Multicolor Imaging of Fixed Cells
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Fixed cells were imaged using a Zeiss LSM980, with a Plan-Achromat 63 × 1.4 NA oil immersion objective (Carl Zeiss, 420782-9900-000). Three laser lines: 405, 488 and 642 nm were used for excitation of Hoechst, Alexa-fluor 488 and Alexa-fluor 647 fluorophores. Built-in multi-band dichroic mirror MBS405/488/561 (Carl Zeiss, 1784-995) were used to reflect excitation laser beams onto samples. For fluorescence signal collection, the used emission spectral bands were: 410–524 nm (Hoechst), 493–578 nm (Alexa-fluor 488) and 564–697 nm (Alexa-fluor 647). The green channel (Alexa-fluor 488) was imaged using a 1 gallium arsenide phosphide (GaAsP) detector, while the blue (Hoechst) and red (Alexa-fluor 647) channels were imaged using two multi-anode photomultiplier tubes (MA-PMTs). For imaging acquisition and rendering, Zeiss ZEN Blue software (v2.3) was used. Confocal Images were deconvolved using the Zeiss ZEN Blue software (v2.3), using the regularised inverse filter method.
5
Microscopy Imaging and Analysis Protocol
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Bright-field images were captured with a PD70-IX2-UCB microscope and CellSens software (Olympus). Fluorescence images were obtained using a Yokogawa CSU-X1 spinning disk scanner coupled to a Zeiss Observer Z1 inverted microscope and controlled with Zen Blue software (Carl Zeiss). Zen Blue software (Carl Zeiss) was used to count cells and measure surface areas. For the quantification of immunofluorescence signals in the brain, the values for six successive slices in each hemisphere and each individual were averaged.
6
Neurogenesis Imaging and Quantification
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All imaging was performed on a Zeiss Axio Observer Z.1 with apotome digital imaging system and Axiocam 506 monochrome camera (Zeiss). Images of cultured NSCs captured using Zen Blue software (Zeiss) were converted to Tag Image File Format (Tiff) for automated cell counting using ImageJ. Tissue sections were imaged using a 20x objective as z-stacks with 20× 1 μm steps. Images were analyzed using Zen Blue software (Zeiss). RGLs and IPCs, EdU+ cells, DCX+BrdU+ cells and NeuN+BrdU+ cells were counted and/or phenotyped by colocalization with cell type identity markers within the SGZ (or SGZ plus GCL for BrdU+ cells). For density measures, the number of cells was divided by the area (in μm2) sampled for counting. Density was then normalized to NT levels within GFP+ or GFP- to allow visualization of GFP+ and GFP- cells on the same y-axis without excessive compression of the less abundant GFP+ cell groups. Imaging and cell quantification were performed while blind to animal identity. 3D reconstructions were performed using 0.5μm z-stacks taken with a 63x oil objective and then rendered with Imaris software (Oxford Instruments). EAAT1 immunolabeling for quantification of knockdown in brain tissue was done using ImageJ to select GFAP+GFP+ area and measure EAAT1 intensity within that area.
7
High-Resolution Confocal Imaging of Organoids
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The histological sections were imaged with the inverted microscope Zeiss Axio Observer.Z1 with confocal unit LSM 800, equipped with solid‐state lasers (405, 561, and 640 nm) and Plan‐Neofluar 10×/0.30 AIR and Plan‐Neofluar 20×/0.50 AIR objectives using zen blue software (Zeiss). Images with 0.329 × 0.329 × 5.500 μm (10×) and 0.156 × 0.156 × 0.700 μm (20×) pixel size were acquired using GaAsP PMT detectors. The acquisition parameters for Alexa Fluor 405, 568, and 647 were: 410–470, 565–617, and 656–700 nm (emission wavelength range) and 1.03 μs (pixel dwell time). The pinhole was set to 1 AU–8.2 μm (10×) and 1 μm (20×). Line average of 2 was applied to all channels.
Cleared whole‐mount organoids were imaged with the inverted microscope Zeiss Axio Observer.Z1 with confocal unit LSM 800, equipped with solid‐state lasers (405, 488, and 561 nm) and Plan‐Neofluar 5×/0.16 AIR and Plan‐Neofluar 10×/0.30 AIR objective usingzen blue software (Zeiss). Images were acquired using GaAsP PMT detectors. The acquisition parameters for Hoechst, GFP, and tdTomato were: 400–486, 486–558, and 575–700 nm (emission wavelength range) and 1 μs (pixel dwell time). The Line average of 2 was applied to both channels. The pinhole was set to 1 AU 32 μm (5×) and 8.2 μm (10×). For z‐stack imaging, slices were acquired with a 16 μm (5×) and 4.1 μm (10×) z‐step size.
Cleared whole‐mount organoids were imaged with the inverted microscope Zeiss Axio Observer.Z1 with confocal unit LSM 800, equipped with solid‐state lasers (405, 488, and 561 nm) and Plan‐Neofluar 5×/0.16 AIR and Plan‐Neofluar 10×/0.30 AIR objective using
8
Multimodal Imaging and Analysis of Neural Structures
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Immunofluorescence whole slides images were acquired with a Thunder Imaging System (Leica-Microsystems) equipped with a 10X dry objective with a NA of 0.45, led excitation, a DFC9000GTC camera and LAS X v3.5 software. Glial cells and neurons images were acquired with a Leica SP5 up-right confocal microscope equipped with a 20X dry objective with a NA of 0.7 and analysed with ImageJ software.
Excitatory and inhibitory synapses images were acquired with a SP8 up-right confocal microscope equipped with a 63X oil immersion objective with a NA of 1.4. Images were taken as z-stacks (five slices, 0,2 μm intervals) with a scan zoom of 2,78X and an image size of 1024 x 1024 pixels (66,38 x 66,38 μm). Synapses quantification was performed in maximal, intensity projection using Synapse Counter plugin for imageJ.53 (link)
Immunohistochemistry whole slides images were acquired with a slide scanner (AxioScan Z1, Zeiss), with a 20X objective and images captured with the Zen Blue Software (Zeiss). An appropriate script was created using Zen Blue Software (additional module for analysis, Zeiss) to analyse each antibody. After training and script optimization, the quantification was run on each image manually, in order to select areas and results exported as excel files with scoring data for each file.
Excitatory and inhibitory synapses images were acquired with a SP8 up-right confocal microscope equipped with a 63X oil immersion objective with a NA of 1.4. Images were taken as z-stacks (five slices, 0,2 μm intervals) with a scan zoom of 2,78X and an image size of 1024 x 1024 pixels (66,38 x 66,38 μm). Synapses quantification was performed in maximal, intensity projection using Synapse Counter plugin for imageJ.53 (link)
Immunohistochemistry whole slides images were acquired with a slide scanner (AxioScan Z1, Zeiss), with a 20X objective and images captured with the Zen Blue Software (Zeiss). An appropriate script was created using Zen Blue Software (additional module for analysis, Zeiss) to analyse each antibody. After training and script optimization, the quantification was run on each image manually, in order to select areas and results exported as excel files with scoring data for each file.
9
Histological Evaluation of Implanted Tissue Constructs
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The constructs and surrounding muscle tissue were retrieved between 2 and 16 weeks after implantation. The samples were washed with PBS 1× and fixed with 4% PFA for 20 min, the fixation solution was removed, and the samples were washed with PBS 1×. Afterward, the samples were incubated ON in 30% sucrose solution at 4°C and then embedded in optimal cutting temperature compound (OCT) (Tissue-Tek) for cryosectioning. For a Masson’s trichrome staining, the samples were cut into 5-μm-thick sections. The slides were initially air-dried and stained with Mayer’s hematoxylin (Sigma-Aldrich) for 5 min, followed by washes with distilled water and trichrome staining (Sigma-Aldrich) for 2 min. Next, the slides were washed twice with 0.2% glacial acetic acid and with double-distilled water. Afterward, the slides were dehydrated by serial immersions in increasing gradient of ethanol and finally dipped in xylene and covered with EUKITT mounting (Sigma-Aldrich). The slides were imaged with the Axio Observer 7 microscope (Ziess). Images were processed using the Zen Blue software (Ziess).
10
Assessment of Hepatic Lipid Accumulation
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The livers were retrieved 16 weeks after implantation and washed with PBS 1×. Then, the samples were fixed with 4% PFA for 20 min, the fixation solution was removed, and the samples were washed with PBS 1×. Afterward, the samples were incubated ON in 30% sucrose solution at 4°C and then embedded in OCT compound (Tissue-Tek) for cryosection. For ORO staining, the samples were cut into 10-μm-thick sections. The slides were dipped in 60% isopropanol and stained with a working solution of ORO (Sigma-Aldrich) in isopropanol (3:2) for 15 min. Then, the slides were washed with distilled water and stained with hematoxylin (Sigma-Aldrich) for 2 min. Next, the slides were washed with tap and distilled water and mounted with Fluoromount-G and covered with coverslips. For histopathological evaluation by a veterinary pathologist, the slides were sent to PATHO-LOGICA Ltd. The ORO-stained area was evaluated by a scoring scale. The slides were imaged with the Axio Observer 7 microscope (Ziess). Images were processed using the Zen Blue software (Ziess).
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