Confocal images of fluorescent specimens were obtained with a Nikon A1 confocal microscope equipped with a 20 × 0.75 numeral aperture (NA) Plan Apo objective and a 60 × 1.49 NA oil immersion objective or a Carl Zeiss LSM 900 confocal microscope equipped with the laser module URGB (diode laser 405 nm; diode laser 488 nm; diode (SHG) laser 561 nm and diode laser 640 nm) and the Airyscan 2. Plan-Apochromat 20x (0.8 NA) objective. Confocal images were captured with a distance interval of 0.5 µm between z-sections. The xy view of the confocal images is presented as maximal projections of z stacks, and xz or yz slice views of the regions of interest were reconstructed to illustrate the protein colocalization. For cell culture, images were acquired by a single focal plane. All confocal images were captured and processed using Nikon NIS-Elements AR (v.4.6) or ZEN 3.0 (Carl Zeiss), whereas brightness, contrast and gamma correction were performed if necessary. Confocal images with Z-stacks were utilized for 3D reconstruction using Imaris 9.7 (Oxford Instruments) (debris engulfment) or ZEN 3.0 (Carl Zeiss) (whole-mount retina).
Zen 3
Manufactured by Zeiss
Sourced in Germany, United States, Japan
ZEN 3.0 is a microscope imaging software developed by Zeiss. It provides a user interface for the control and operation of Zeiss microscopes and the acquisition, processing, and analysis of microscope images.
Automatically generated - may contain errors
Lab products found in correlation
Axio observer, by Zeiss (7 mentions)
Lsm 900, by Zeiss (6 mentions)
Lsm 900 confocal microscope, by Zeiss (5 mentions)
Lsm 710, by Zeiss (5 mentions)
Axio scan z1, by Zeiss (5 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (4 mentions)
Lsm 780, by Zeiss (4 mentions)
Triton x 100, by Merck Group (4 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (4 mentions)
Prism 9, by GraphPad (4 mentions)
Axio imager z2 microscope, by Zeiss (4 mentions)
Imagej, by Zeiss (3 mentions)
Hoechst 33342, by Thermo Fisher Scientific (3 mentions)
Lsm 800, by Zeiss (3 mentions)
Axio imager m2, by Zeiss (3 mentions)
Lsm 880, by Zeiss (3 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (3 mentions)
Lsm 880 confocal microscope, by Zeiss (3 mentions)
Axio observer 7, by Zeiss (3 mentions)
Axiocam 506, by Zeiss (3 mentions)
177 protocols using zen 3
1
Confocal Imaging of Fluorescent Specimens
2
Visualizing T. gondii Infection in HFF Cells
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HFF cells were grown to confluency on glass coverslips and infected with T. gondii parasites expressing previously mentioned epitope tags. After time periods ranging between 18 and 36 h, infected coverslips were fixed using 3.7% formaldehyde and processed for indirect immunofluorescence assay (IFA) as described previously [87 (link)]. Primary antibodies were detected by species-specific secondary antibodies conjugated to Alexa594/488. Coverslips were mounted in Vectashield (Vector Labs) and viewed with an Axio Imager.Z1 fluorescence microscope (Zeiss). Images were processed with the ZEN 3.7 software (Zeiss), which included deconvolution and adaptation of the magenta pseudocolor from the 594 fluorophores. The Pearson’s correlation coefficient was calculated using ZEN 3.7 software (Zeiss) [41 (link)].
For western blotting, parasites were lysed in 1× Laemmli sample buffer (50 mM Tris-HCl [pH 6.8], 10% glycerol, 2% SDS, 100 mM DTT, 0.1% bromophenol blue) and boiled at 100°C for 10 min. Lysates were resolved by SDS-PAGE and transferred to nitrocellulose membranes. Protein blots were probed with the appropriate primary antibody followed by the corresponding secondary antibodies conjugated to horse radish peroxidase (HRP). Proteins were visualized by chemiluminescence (Thermo Scientific) and imaged on ChemiDoc XRS+ (Bio-Rad).
For western blotting, parasites were lysed in 1× Laemmli sample buffer (50 mM Tris-HCl [pH 6.8], 10% glycerol, 2% SDS, 100 mM DTT, 0.1% bromophenol blue) and boiled at 100°C for 10 min. Lysates were resolved by SDS-PAGE and transferred to nitrocellulose membranes. Protein blots were probed with the appropriate primary antibody followed by the corresponding secondary antibodies conjugated to horse radish peroxidase (HRP). Proteins were visualized by chemiluminescence (Thermo Scientific) and imaged on ChemiDoc XRS+ (Bio-Rad).
3
Automated Bacterial Detection and Quantification
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For manual image viewing and analysis, Fiji (ImageJ v1.53t, [49 (link)]) and Zen 3.4 (blue edition, Carl Zeiss) were used. Stitching of individual tiles was performed using Zen 3.4 (blue edition, Carl Zeiss) or the Fiji Stitching Plugin [50 (link)]. Automated detection and counting of the bacterial regions across a voxel were employed using a novel in-house algorithm scripted in Python language [51 ].
4
Quantitative Histomorphometric Analysis of Scaffold
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In addition to the semi-quantitative analysis, the thick sections from the scaffold center were quantitatively analyzed by histomorphometry. For this purpose, images of the cross sections (Zeiss Axio Imager 2, Carl Zeiss Microscopy GmbH, Jena, Germany) were taken at ×20 magnification using the Zeiss Axio Cam Mrc digital camera and the software Zeiss ZEN 3.0 (Carl Zeiss Microscopy GmbH, Jena, Germany). The images were evaluated using the software Zeiss ZEN 3.0 (Carl Zeiss Microscopy GmbH, Jena, Germany). The percentage area of scaffold material, ingrown bone tissue, and soft tissue (granulation tissue, bone marrow) was measured within a predefined circle (Ø = 1,060 pixels ≙ diameter of the OR of the semiquantitative examination (Ø = 4.24 mm) ≙ scaffold diameter), which was placed centrally around the initial implantation area.
5
Metabolite effects on Candida albicans filamentation
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C. albicans (1 × 104 cell/ml) in KBM with or without supplementation of individual metabolites or in combination was incubated for 24 h at 37 °C with 5% CO2 inside the Cell Discoverer 7 microscope (Zeiss), in which a bright field picture was taken every hour, images were exported with Zeiss Zen3.1 (blue edition). Hyphal length was measured at 4 h images using Zeiss Zen3.1 (blue edition) Table 2 .
Tested metabolites to impair C. albicans filamentation.
| Compound name | Experimental concentration | Company | |
|---|---|---|---|
| alone | in combination | ||
| 2-deoxyinosine | 50 mM | – | Sigma |
| Allantoine | 50 mM | – | Roth |
| Alpha hydroxycaproate (HICA) | 50 mM | 5 mM | Sigma |
| Cytosine | 50 mM | 5 mM | Sigma |
| Histidine | 50 mM | – | Roth |
| Hydroxymethylbutyrate | 50 mM | 5 mM | Sigma |
| Indolelactate | 50 mM | 5 mM | Sigma |
| Sodium lactate | 50 mM | 15 mM | Sigma |
| Phenyllactic acid | 50 mM | 5 mM | Sigma |
| Phenylpyruvate | 50 mM | 5 mM | Sigma |
| Pipecolate | 50 mM | – | Sigma |
| Thymine | 50 mM | – | Sigma |
| Uridine | 50 mM | – | Roth |
6
Histological Analysis of Organ Tissues
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The preserved tissues (kidney, liver and heart) were macroscopically examined and then dehydrated in graded ethanol and xylene baths. The dehydrated sections (measuring 3–4 µm) were then embedded in paraffin wax and stained with hematoxylin and eosin (H-E). The liver, heart and kidney tissue structures were examined using an Axiolab A5 light microscope with Axiocam 208 color and ZEN 3.0 software (Zeiss, Jena, Germany). Microscopic evaluation was performed at 10× and 40× magnification. Morphometric measurements of 5 arcuate arteries and 5 arterioles were performed for each individual. Four measurements were made at the ×40 lens magnification using the ZEN 3.0 software (Zeiss, Jena, Germany) for each type of vessel.
7
Live Imaging of Axonal Retraction in NPs
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Live Imaging analysis was performed at CEINGE Advanced Light Microscopy Facility using the automated platform Celldiscoverer7 system (Zeiss, Oberkochen, Germany) equipped with a heated stage (37 °C and 5% CO2) and an Orca flash 4.0 camera (Hamamatsu). Briefly, timelapse of 6 well plates containing NP exposed to Sema 3A, GFP and Ctrl media were acquired using a Plan Apochromat 20x/0.7 objective and 1 × tubelens. Images (24 frames) were captured at 5 min intervals (Zeiss, Oberkochen, Germany) in phase gradient contrast. Axonal retraction was quantified by manual measuring of the distance between the neuronal soma and the axon edge, at time 0 and after 60 min of exposure to Sema 3A or control media, using the Zen 3.1 Software (Zeiss, Oberkochen, Germany).
All materials used for cell culture, WB, IF and live imaging experiments are reported in additional files (see Additional File1 : Table S1).
All materials used for cell culture, WB, IF and live imaging experiments are reported in additional files (see Additional File
8
Intravital Imaging using Light Sheet Microscopy
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Intravital imaging was performed with a Light sheet microscope (Z1, Zeiss) enabled for dual-side illumination. It was equipped with a 20 x detection objective (W Plan-Apochromat, numerical aperture of 1.0) and an sCMOS pco. edge 4.2 camera. Furthermore, the setup was temperature-controlled by the TempModule S1 and the TempModuleCZ-LSFM in combination with the PeltierBlock S and a Temperature Sensor, which were both assembled with the sample chamber. This allowed imaging at the optimal temperature for the fish (26 °C). Image processing, including dual side fusion, brightness/contrast adjustment, and image export was conducted using the Zen 3.1 software (blue edition, Zeiss). The 3Dxl rendering module (powered by arivis) was employed for three-dimensional reconstructions of the recorded z-stacks. Conversion of z-stacks into movies was performed using the arivis Vision4D software (arivis AG).
9
Microtubule Dynamics in Algal Cells
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overnight cultures were resuspended in fresh TAP or M1 (CC-1690) the next morning. Cells were treated with either DMSO, paclitaxel (PTX, Invitrogen P3456), Colchicine, or LiCl, fixed in equal amounts of 2% Glutaraldehyde, and then imaged on a Zeiss Axioscope 5 DIC with 40x magnification and Zeiss Zen 3.1 software.
10
Histological Detection of Amyloid Deposits
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Thioflavin S and Congo Red are two major histological stains used to detect any form of amyloid. The stainings were performed according to the protocol used for routine clinical diagnostics at the Department of Neuropathology at University Hospital Aachen, Germany. For Thioflavin S staining, hydrated paraffin sections were stained for 5 min in Mayer′s hemalum solution, washed with water for 5 min and then stained with 1% Thioflavin S solution (w/v in ddH2O; Sigma, Munich, Germany) for 5 min. Staining was differentiated in 70% ethanol, rinsed in ddH2O and mounted in glycerol-gelatin. Thioflavin S bound to amyloid emits green fluorescence.
For Congo Red staining, hydrated paraffin sections were stained for 10 min in Mayer′s hemalum solution, washed with water for 10 min, incubated in fresh 1% NaOH solution (w/v in 80% ethanol, Merck, Darmstadt, Germany) for 20 min and stained with 0.5% Congo Red solution (w/v in 80% ethanol/0.1% NaOH, Merck, Darmstadt, Germany) for 20 min. Staining was differentiated in 100% ethanol, and mounted in vitroclud. Histological sections were viewed and imaged with an Axio Scope.A1 microscope (Zeiss, Oberkochen, Germany) using ZEN 3.1 software (Zeiss, Oberkochen, Germany).
For Congo Red staining, hydrated paraffin sections were stained for 10 min in Mayer′s hemalum solution, washed with water for 10 min, incubated in fresh 1% NaOH solution (w/v in 80% ethanol, Merck, Darmstadt, Germany) for 20 min and stained with 0.5% Congo Red solution (w/v in 80% ethanol/0.1% NaOH, Merck, Darmstadt, Germany) for 20 min. Staining was differentiated in 100% ethanol, and mounted in vitroclud. Histological sections were viewed and imaged with an Axio Scope.A1 microscope (Zeiss, Oberkochen, Germany) using ZEN 3.1 software (Zeiss, Oberkochen, Germany).
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