All slides were imaged on the Zeiss 710 Inverted Laser Scanning Confocal Microscope (Duke University Light Microscopy Core Facility) at full 25 μm depth as Z-stacks. For quantifications, 2 sections per animal were imaged, and a 2 × 2 grid tile scan was performed using the 20× objective centered on the VH of the SC, totaling 8 fields per mouse. Following quantification, these replicates were averaged to generate a single n for statistical analyses. Semiautomated quantification was conducted using the Imaris for Neuroscientists Cell Imaging Software ver. 9.3.0. (Bitplane) unless otherwise indicated. Briefly, the Surfaces tool was used to identify either ASC specks/strings or cells, and intensity thresholds of counterstain signals within the surfaces were used to quantify the desired characteristics. ASC specks and ASC strings counts in Figure 1 were manually enumerated using the LSM Browser software (Zeiss).
Lsm browser software
Manufactured by Zeiss
The LSM Browser software is a tool developed by Zeiss to allow users to view and analyze data generated by Zeiss laser scanning microscopes. The software provides a user-friendly interface for accessing and managing microscope data files.
Automatically generated - may contain errors
Lab products found in correlation
Axiovert 200, by Zeiss (2 mentions)
Lsm 510 microscope, by Zeiss (2 mentions)
Colcemid, by Merck Group (1 mentions)
Plan apochromat 100 1.4, by Zeiss (1 mentions)
S1 nuclease, by Thermo Fisher Scientific (1 mentions)
Metamorph, by Universal Imaging (1 mentions)
Mab3418, by Merck Group (1 mentions)
Lsm 510 confocal laser scanning microscope, by Zeiss (1 mentions)
Bromodeoxyuridine (brdu), by Merck Group (1 mentions)
Nocodazole, by Merck Group (1 mentions)
Fluoroshield, by Merck Group (1 mentions)
Coverslip, by Merck Group (1 mentions)
Plan apochromat 63, by Zeiss (1 mentions)
11 protocols using lsm browser software
1
Quantitative Analysis of ASC Specks and Strings in Mouse Spinal Cord
2
Chromosomal Aberration Analysis Protocol
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Chromosome aberrations were restricted to macrochromosome variation using a method described previously (23 (link)). Briefly cells were treated with CisPt (2 μM, 14 h) followed by 1% colcemid (Sigma) for 2 h. Cells were harvested and hypotonically swollen with 0.9% sodium citrate for 15 min at room temperature, and fixed with freshly prepared 5 ml of methanol:acetic acid (3:1). Fixed cells were dropped onto chilled glass slides, immediately flame, dried, and stained with DAPI. Images were captured in a Zeiss LSM 510 META inverted Axiovert 200M laser scan microscope with a Plan-Apochromat 100× 1.4-numerical- aperature oil immersion differential interference contrast objective lens. Images were captured with a CCD camera and analyzed using LSM Browser software (Zeiss).
3
DNA Fiber Assay for Replication Forks
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The progression of replication forks upon UV exposure was evaluated by a DNA fiber assay, with a 20-min pulse of chlorodeoxyuridine (CldU, Sigma-Aldrich) before UVC irradiation and a 60-min pulse of iododeoxyuridine (IdU) afterward (24 (link)). For experiments with cells expressing photolyases, upon UV irradiation, the cells were incubated with 200 μM IdU in PBS+ supplemented with 5% FBS for 60 min at RT on the photoreactivation apparatus. For experiments with the ssDNA-specific S1 endonuclease, after an IdU pulse, the cells were treated with CSK100 buffer (100 mM NaCl, 300 mM sucrose, 3 mM MgCl2, 10 mM MOPS, 0.5% Triton X-100) for 10 min at RT, then incubated with S1 nuclease buffer (50 mM NaCl, 30 mM sodium acetate pH 4.6, 10 mM zinc acetate and 5% glycerol) with or without S1 nuclease (Invitrogen, Life Technologies) at 20 U/ml for 30 min at 37ºC. See Supplementary Material for more details. DNA fibers were imaged using a fluorescent microscope (Axiovert 200, Zeiss, Jena, Germany) at a magnification of 1000×. Analyses were performed using Zeiss LSM Browser Software. All experiments were performed at least twice independently, and at least 100 fibers were counted for each slide. Results repeated in separate figures represent independent experiments.
4
Quantifying T Cell Cytoskeletal Dynamics
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The intensity of CD11a and F-actin stainings were analysed using the Linescan Function of software MetaMorph (Universal Imaging–Bedford Hills, NY, USA). A line of reference of ∼0.5 μm was drawn at the region of interest. This line was drawn inside the T cell to exclude fluorescence originating from the target. The software calculates the average intensity along this line of reference for 12 pixels of width. The regions were defined as shown in Figs 3 , 6 and 7 . The intensities were measured at the ‘synapse centre' and in ‘peripheral synapse' regions of one T cell, using the same line of reference. To estimate the adhesion status of T cells, each cell was considered as a region, carefully drawn around the IRM images. Each region was analysed for area and brightness (MetaMorph Software). The polarization of the MTOC of the T cell towards its target was evaluated by measuring the distance between MTOC and the centre of the T-cell-target contact zone, using the profile function of the Zeiss LSM Browser software.
5
Quantifying Neuronal Internalization of Fluorescent Peptides
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Following conjugation of each DPR to FITC, labeled DPRs were applied to primary rodent cortical neurons on DIV4 at a final concentration of 37.5 μg/ml. The cells were subjected to immunocytochemistry, as described below, using primary antibodies against MAP2 (dilution 1:500, Millipore, MAB3418, mouse monoclonal, clone AP20) and secondary anti-mouse Cy5-conjugated antibodies (dilution 1:250, Jackson Immunoresearch, 115-175-146, Goat whole IgG). The cells were then imaged using a Zeiss LSM510 laser scanning confocal microscope. For micrographs of neurons treated with FITC-labeled peptides, 20 optical slices were taken for each cell with a step size of 5 nm between slices. Z-sections were constructed using LSM Browser software (Zeiss). A total of > 200 neurons were counted from each population to determine the frequency of internalization.
6
Quantifying Stromule Dynamics in Plants
7
Detecting PRR Tracts in Human Cells
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PRR tract detection in human cells was adapted from Daigaku et al., who first performed the technique in budding yeast (27 (link)). Briefly, upon UV irradiation the cells received medium with 100 ng/ml of nocodazole (Sigma-Aldrich) for 24 h. For the last 4 h, 10 μM BrdU (Sigma-Aldrich) was added to the medium. Glass slides were prepared as for the DNA fiber assay (described above). ssDNA was stained with mouse anti-ssDNA antibody (Millipore) and anti-mouse Alexa Fluor 594, and BrdU was detected with rat anti-BrdU and anti-rat Alexa Fluor 488. The slides were mounted using Fluoroshield (Sigma-Aldrich) and DNA fibers were imaged using a fluorescent microscope (Axiovert 200, Zeiss) at a magnification of 1000×. Analyses were performed using Zeiss LSM Browser Software. All experiments were performed twice independently. For quantification, only ssDNA with no continuous BrdU staining and with at least one clear and distinguishable BrdU patch were evaluated. The lengths of ssDNA were converted into kilobases using the conversion factor 1 μm = 2.59 kb (27 (link)), and at least ten independent fibers were evaluated per condition and per experiment. Each figure represents different and independent experiments, performed simultaneously for proper comparison.
8
Live Imaging of Pupal Development
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For live imaging, pupae were mounted in halocarbon oil (series 700; Halocarbon Products) on a coverslip (Sigma) and imaged with a laser 510 scanning confocal microscope (ZEISS) at 25°C with a 40 × objective, using LSM Browser software (ZEISS). Z-stacks had a 3 μm step size. Pupal collection and staging were done as described by [27 ].
9
Imaging G4 DNA Structures in FANCJ-Deficient Cells
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The isogenic fancj−/− transfected cell lines were treated with DMSO or 5 μM TMS for 6 h. Cells were fixed with freshly prepared 4% formaldehyde and stained for immunoreactive G4 using a mouse anti-G4 1H6 monoclonal antibody (1:1000, Millipore) previously described (22 (link)). Immunofluorescence analyses were performed with a Zeiss LSM 510 META inverted Axiovert 200M laser scan microscope with a Plan-Apochromat 63× 1.4-numerical- aperature oil immersion differential interference contrast objective lens. Images were captured with a CCD camera and analyzed using LSM Browser software (Zeiss).
10
Confocal Imaging of Adult Brains
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Confocal serial scanning images were acquired at 1.5-2 µm intervals using a LSM 510 microscope (Zeiss) using either a 20×0.5 Plan-Neofluar or a 40×1.4 Plan-Neofluar objective (Zeiss). Stacks were processed using the Zeiss LSM Browser software and whole or parts of stacks were visualized as maximum intensity projections. Brightness, contrast, size and resolution of the images were processed in Adobe Photoshop CS. The movie of the G10011 adult brain was generated with ImageJ software (Movie 1 ).
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