DLD-1 cells were sequentially labelled with 10 mM IdU (Sigma-Aldrich) and 100 mM CldU (Sigma-Aldrich) for 30 min each. After labelling, cells were placed on ice immediately to stop DNA replication and subsequently centrifuged (300g, 5 min at 4 °C). After washing three times in PBS, 1 × 106 cells were placed onto a microscope slide and incubated with the spreading buffer (200 mM Tris-HCl pH 7.5, 0.5% SDS and 50 mM EDTA) for 1 min. The slides were tilted 15° to extend the DNA fibres. After fixation using methanol/acetic acid (3:1), the DNA was denatured using 2.5 M HCl and blocked with 1% BSA for 2 h before staining with primary (rat anti-BrdU for CldU and mouse anti-IdU) and secondary antibodies conjugated with Alexa Fluor 488 or 546. Images were acquired using a confocal microscope (Lecia TCS SP8) and analysed using the ZEN 2.3 SP1 (ZEISS) software. Statistical analysis was performed using Prism 8 (GraphPad software).
Zen 2.3 sp1
Manufactured by Zeiss
Sourced in Germany
Zen 2.3 SP1 is a software suite developed by Zeiss for microscopy imaging and analysis. It provides a comprehensive set of tools for image acquisition, processing, and visualization.
Automatically generated - may contain errors
Lab products found in correlation
Prism 7, by GraphPad (2 mentions)
Prism 9, by GraphPad (2 mentions)
Image lab, by Bio-Rad (2 mentions)
Lsm 800, by Zeiss (2 mentions)
Lsm 710, by Zeiss (2 mentions)
Lsm 780, by Zeiss (2 mentions)
Lsm 880, by Zeiss (2 mentions)
Tcs sp8, by Leica (1 mentions)
Prism 8, by GraphPad (1 mentions)
Confocal microscope lsm880, by Zeiss (1 mentions)
Plan apochromat 63x 1.4 oil dic m27, by Zeiss (1 mentions)
Zen 2011 sp3, by Zeiss (1 mentions)
Scaffold 4, by Proteome Software (1 mentions)
Lsm 5 exciter, by Zeiss (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Zen black, by Zeiss (1 mentions)
Sp5 confocal microscope, by Leica (1 mentions)
Vectashield, by Vector Laboratories (1 mentions)
14 protocols using zen 2.3 sp1
1
Dual DNA Replication Labeling Assay
2
Tracking Endosomal Escape of TPPA/siRNA Complexes
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HCEC cells were seeded into 35 mm dishes at a density of 2 × 105 cells per well and incubated for 24 h. Then the cells were transfected with TPPA/Cy5-siRNA (15:1, 50 nM) was. After 4 h, cells were washed 3 times with 1 × PBS and treated with100 μL DMEM containing 1 μL Hoechst 33342 (1 mg/mL in PBS, for staining nuclei) and 0.3 μL LysoTracker Green (1:3000 in PBS, for staining endosomes and lysosome). After 30 min of staining, cells were washed twice with 1 × PBS and then recorded with a Zeiss confocal microscope (LSM880, Carl Zeiss, Germany). To analyze the endosomal escape time of PPA/siRNA, cells were stained and imaged according to the above protocol at 4, 6, 10, and 24 h post-transfection, respectively. Images were analyzed using ZEN (ZEN2.3 SP1, Carl Zeiss, Germany).
3
Quantitative Image Analysis Methods
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Immunoblotting plots were digitally scanned by a ChemDoc Imaging System. Band intensity was measured by Image Lab (BioRad). Imaging data were processed by either Zen 2.3 SP1 (Zeiss) or by Nikon Elements (Nikon). Fluorescence intensity was measured by Imaged J or by Imaris in the case of 3D imaging. Unless specified, the n values in the figure legend indicate the number of independent biological replicates. Statistically analyses were performed using GraphPad Prism 9. For two group comparison, unpaired Student’s t-test was used. For multi-group comparison, we used one-way ANOVA. For comparing life span in flies, Gehan-Breslow-Wilcoxon test was used. Error bars, shown as mean ± SD and p values were calculated by GraphPad Prism 9. A value of p < 0.05 was considered as statistically significant. Graphs were prepared with GraphPad Prism 7.
4
Airyscan Imaging of Transcription Sites
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All Airyscan images were acquired using a Zeiss Plan-Apochromat 63x/1.4 Oil DIC M27 objective due to its well-characterized point spread function. First an embryo at the appropriate developmental stage (stage 15 for most embryos) and proper orientation was located. The band of mRNA expression in high Ubx regions of the first abdominal (A1) segment was then found. Within that band, areas containing transcription sites in nuclei of high Ubx expression were imaged. Images with both Ubx and Hth were acquired in the same manner by locating the proper area using the mRNA and Ubx. When Ubx was imaged together with RNA polymerase II, a histone marker, or other transcription factors, Ubx expression levels were used to locate the region of interest.
The optimal setting suggested by Zeiss for the number of pixels in the x-y direction (40 nm per pixel) and displacement in the z-stack (190 nm) were used for all Airyscan images. The images from different fluorophores were acquired sequentially with the appropriate laser lines (405 nm, 488 nm, 561 nm, or 633 nm) and spectral filters. The laser power and gain were adjusted to maximize the signal to noise ratio within the dynamic range of the Airyscan detector. The acquired stacks were processed with Zen 2.3 SP1 (Carl Zeiss Microscopy GmbH, Jena, Germany) in 3D mode to obtain super-resolved images.
The optimal setting suggested by Zeiss for the number of pixels in the x-y direction (40 nm per pixel) and displacement in the z-stack (190 nm) were used for all Airyscan images. The images from different fluorophores were acquired sequentially with the appropriate laser lines (405 nm, 488 nm, 561 nm, or 633 nm) and spectral filters. The laser power and gain were adjusted to maximize the signal to noise ratio within the dynamic range of the Airyscan detector. The acquired stacks were processed with Zen 2.3 SP1 (Carl Zeiss Microscopy GmbH, Jena, Germany) in 3D mode to obtain super-resolved images.
5
Localization of LjCSyGT Fusion Proteins
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LjCSyGT stop-less cDNA was cloned into pDONR™221 using the primer pairs, 23 and 24’. LjCSyGT cDNAs were transferred from pDONR entry clones to the pK7WGR2 or pK7RWG253 (link) vector through an LR clonase reaction, generating plasmids no. 22 and 23. Hairy roots transformed with RFP-LjCSyGT and LjCSyGT-RFP were generated using Agrobacterium rhizogenes AR1193, as described52 . The plasmids ER-gk and G-gk were used as ER system and Golgi apparatus markers, respectively54 (link). The localisation of RFP-LjCSyGT or LjCSyGT-RFP was examined with a confocal microscope (LSM710, Carl Zeiss) using an EC Plan-Neofluar objective lens. RFP fluorescence was excited at 543 nm and emission was detected at 548–680 nm. GFP fluorescence was excited at 488 nm and emission was detected at 493–538 nm. Microscopic images were taken with LSM710 (Carl Zeiss) and ZEN2011 SP3 (Carl Zeiss) and analysed with ZEN2.3 SP1 (Carl Zeiss).
Hairy roots of mutant lines transformed with either RFP-LjCSyGT or LjCSyGT-RFP were harvested and extracted as described previously for LC–MS analysis. Two individual transformants with the same construct were combined to obtain sufficient sample.
Hairy roots of mutant lines transformed with either RFP-LjCSyGT or LjCSyGT-RFP were harvested and extracted as described previously for LC–MS analysis. Two individual transformants with the same construct were combined to obtain sufficient sample.
6
Visualizing CFSE-labeled Microvesicles
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CFSE-labeled MVs were added in 8 well μ-slides, ibiTreat (Ibidi, Martinsried, Germany). Following a short centrifuge of the slide, MVs were immediately visualized using an LSM 780 confocal laser-scanning microscope driven by Zen 2012 software. 1 μm latex beads were also visualized for comparison. Images were processed using the software Zen2.3SP1 (Carl Zeiss).
7
Quantitative Image Analysis Methods
8
Quantitative Protein Analysis Pipeline
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Confocal images were analyzed with a Zen 2.3 SP1 (Zeiss, Oberkochen, Germany). Flow cytometry data were analyzed with FlowJo 10 (FlowJO LLC, Ashland, OR, USA). Mass spectroscopy (MS) data were analyzed with Scaffold4, (Proteome Software, Portland, OR, USA). In silico determination of protein identities was performed with the online database and tools of UniProt (European Bioinfirmatics Institue, Swiss Institue of Bioinformatics and Protein Information Ressource). Protein polyacrylamide gel electrophoresis pictures were acquired and processed using FusionCapt version 17.03 (Witec, Sursee, Switzerland).
9
Fluorescent Labeling of Tal CBM and RBP
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Labelling assays were performed as previously described (Dieterle, et al., 2017 ) with the following modifications; for all labelling assays, LM17 was supplemented with 10 mM CaCl2 before fluorescently tagged STP1 Tal CBM and RBP module were added to 300 µl of cells in the early exponential phase (OD600nm 0.3–0.4) at a final concentration of 5, 10, 20, 50 or 100 µg/ ml. In the case of competitive inhibition assays, both fluorescently tagged Tal CBM and RBP module were added at a final concentration of 100 µg/ ml each. Incubation took place at 37 °C for 12.5 min before washing in 120 µl of SM buffer (50 mM Tris‐HCl pH 7.5, 100 mM NaCl, 10 mM MgSO4). Fluorescent labelling of cells was visualized using an LSM 5 Exciter (Zeiss, Germany). Final images were analysed using the Zen 2.3 SP1 software package (Zeiss, Germany).
10
Trachea Imaging with Confocal Microscopy
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