For the ciliogenesis assay, mIMCD3 cells were grown on glass coverslips for 6 days. After methanol fixation, the cells were stained with the primary antibodies rabbit anti-NME3 (Proteintech, 15136-1-AP; 1:100), -acetylated tubulin (Sigma-Aldrich, T6793; 1:2000), and -γ-tubulin (Sigma-Aldrich, T6557; 1:200) and with Hoechst 33342. Antibodies were visualized using Cy3- or Alexa Fluor–488-labeled secondary antibodies at a dilution of 1:500 (Jackson ImmunoResearch Laboratories). Confocal microscopy was done using the LSM510 Duo-Live microscope equipped with an LCI Plan-Neofluar 63×/1.3 Imm Korr DIC (differential interference contrast) objective (Carl Zeiss). ZEN-Black 2010 software (Carl Zeiss) was used for image acquisition. z-stacks were taken to include all cilia in different z-positions and then projected to one plane (maximum intensity projection). To study three-dimensional (3D) protein distributions, z-stacks were converted to 3D images in Imaris 7.6 (Bitplane).
Zen black 2010
Manufactured by Zeiss
Zen Black 2010 is a software application developed by Zeiss for the operation and control of microscopy systems. It provides a user-friendly interface for managing the hardware components, image acquisition, and data processing tasks associated with microscopy workflows.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 780, by Zeiss (4 mentions)
Zen blue 2.3 lite software, by Zeiss (2 mentions)
Acetylated tubulin, by Merck Group (1 mentions)
Alexa fluor 488 labeled secondary antibody, by Jackson ImmunoResearch (1 mentions)
γ tubulin, by Merck Group (1 mentions)
Cyanine3 (cy3), by Jackson ImmunoResearch (1 mentions)
Fluorodish, by World Precision Instruments (1 mentions)
Mouse anti gfp, by Takara Bio (1 mentions)
6 protocols using zen black 2010
1
Ciliogenesis Visualization in mIMCD3 Cells
2
Super-resolution Imaging of Drosophila NMJs
3
Super-resolution Imaging of Drosophila NMJs
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Super-resolution imaging was performed on a Carl Zeiss Elyra PS1 inverted microscope using a Plan-Apo 100X (1.46 NA) oil immersion objective and an EM-CCD Andor iXon 885 camera. We collected 3 5 phases at 3 3 angles for a total of 15 images per plane. Singles NMJ 6/7 at the A3 segment were captured by multiple frames (3–4 per NMJ); the stacks of z sections were taken at a spacing of every 100 nm. All raw images were processed and reconstructed in 3D using Zen Black 2010 software (Carl Zeiss). The images were also channel aligned using an alignment matrix generated by imaging colored beads. The PSD areas were estimated using the Fiji distribution algorithm (ImageJ) (Schindelin et al., 2012 (link)). Single ROIs corresponding to the maximum PSD areas were selected and measured using either the wand tool (with legacy and regulated tolerance) or manually, for overlapping regions. At least 1400 single PSDs from 12 or more different NMJs for each genotype. Statistical analyses were performed with Prism using one-way ANOVA with Tukey’s post hoc test for multiple comparison, frequency distribution and cumulative distribution. Data are presented as mean ± SEM.
4
Quantifying TRPM8-Rap1 Protein Interactions
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Proximity Ligation Assays (PLA) were performed using Duolink (R) In Situ Red-Starter Kit Goat/Rabbit (Sigma-Aldrich, Saint-Quentin Fallavier, France). PC3 cells were seeded (10 × 104 cells/dish) on confocal FluoroDish (World Precision Instruments) and then transfected or not with TRPM8 wt or E207A Y240A and Rap1 N17 (1 μg for each plasmid). After fixation, cells were permeabilized with 4% platelet-activating factor for 10 min and then incubated in the blocking buffer (PBS 4% BSA) for 1 h and 30 min at room temperature. Primary antibodies diluted in the antibody diluents (rabbit anti–TRPM8 antibody, N571644, 1:200; Antibodies-online; mouse anti-GFP 1:200; Takara) were then added to the cells overnight at 4 °C in a humidified chamber. The rest of the protocol was performed according to the manufacturer’s instructions. Recordings were performed by confocal imaging (LSM880; ZEISS) using z-stack superposition (Zen black 2010 software; ZEISS). Appropriate controls were performed by incubating PC3 cells with both primary antibodies separately. In order to assess where the interaction between the two proteins occurs, ER staining was assessed 24 h time after transfecting the cells with 0.3 µg of Ds-Red2 plasmid fused to the ER targeting sequence of calreticulin (Ds-Red2 ER probe, Takara Bio Europe SAS, Saint-Germain-en-Laye, France) in bottom glass dishes.
5
Confocal Imaging of Transgenic Roots
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Immunostained root sections were imaged on a confocal microscope (Zeiss LSM 780).
Autofluorescence observation was performed using an argon laser at 405 nm and secondary antibodies were excited at 561 nm. Both were detected at a 566-679 nm window using the same settings (gain, offset and resolution which were manually set up) to allow quantification measurements (detailed in the Supplemental Methods S1).
For LaPG3::nlsYFP, DR5::nlsYFP and screening of transformed cluster roots, mCherry internal control and nlsYFP were excited at 561 nm and 514 nm respectively and detected at 583-696 nm and 519-583 nm windows, respectively. Observations were made using Plan-Apochromat 10x/0.45 M27 and 20x/0.8 M27 objectives. Image acquisition was performed with the Zeiss ZEN black 2010 software and image analysis was conducted using the ZEN blue 2.3 lite software (Carl Zeiss Microscopy).
Autofluorescence observation was performed using an argon laser at 405 nm and secondary antibodies were excited at 561 nm. Both were detected at a 566-679 nm window using the same settings (gain, offset and resolution which were manually set up) to allow quantification measurements (detailed in the Supplemental Methods S1).
For LaPG3::nlsYFP, DR5::nlsYFP and screening of transformed cluster roots, mCherry internal control and nlsYFP were excited at 561 nm and 514 nm respectively and detected at 583-696 nm and 519-583 nm windows, respectively. Observations were made using Plan-Apochromat 10x/0.45 M27 and 20x/0.8 M27 objectives. Image acquisition was performed with the Zeiss ZEN black 2010 software and image analysis was conducted using the ZEN blue 2.3 lite software (Carl Zeiss Microscopy).
6
Confocal Imaging of Transformed Plant Roots
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Immunostained root sections were imaged on a confocal microscope (Zeiss LSM 780). Autofluorescence observation was performed using an argon laser at 405 nm and secondary antibodies were excited at 561 nm. Both were detected at a 566-679 nm window using the same settings (gain, offset, resolution) to allow quantification measurements. For LaPG3::nlsYFP, DR5::nlsYFP and screening of transformed cluster roots, mCherry internal control and nlsYFP were excited at 561 nm and 514 nm respectively and detected at 583-696 nm and 519-583 nm windows, respectively. Observations were made using Plan-Apochromat 10x/0.45 M27 and 20x/0.8 M27 objectives. Image acquisition was performed with the Zeiss ZEN black 2010 software and image analysis was conducted using the ZEN blue 2.3 lite software (Carl Zeiss Microscopy).
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