Nuclei were isolated from iPSNs and postmortem human motor and occipital cortex tissue using the Nuclei Pure Prep Nuclei Isolation Kit (Sigma Aldrich) following manufacturer protocol with slight modifications as previously described [4 , 5 (link)]. About 10 million iPSNs or 100 mg of frozen postmortem motor cortex tissue (obtained from the Target ALS Human Postmortem Tissue Core (see Additional file 2 : Table 2 for demographic information) was used for nuclei isolation. A 1.85 M sucrose gradient was used to enrich for neuronal nuclei. Following isolation, nuclei were centrifuged onto collagen coated (1 mg/mL; Advanced Biomatrix) slides with a CytoSpin 4 centrifuge (Thermo Fisher Scientific) and immunostained as previously described [4 , 5 (link)] (see Additional file 2 : Table 3 for antibody information). Isolated nuclei were subsequently imaged by super resolution structured illumination microscopy (SIM) using a Zeiss ELYRA S1 as previously described [4 , 5 (link)]. All images were acquired using identical imaging parameters (e.g. laser power, gain) and subjected to default SIM deconvolution and processing in Zeiss Zen Black 2.3 SP1. Representative images are presented as 3D maximum intensity projections generated in Zeiss Zen Black 2.3 SP1. Images were faux colored green for contrast and display.
Zen black 2.3 sp1
Manufactured by Zeiss
Zen Black 2.3 SP1 is a software platform developed by Zeiss for the control and configuration of microscope systems. The software provides a user-friendly interface for managing various microscope functions, image acquisition, and data processing.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 880 confocal microscope, by Zeiss (10 mentions)
Bz x700 microscope, by Keyence (3 mentions)
Bz x analyzer, by Keyence (3 mentions)
Illustrator cc 2018, by Adobe (2 mentions)
Cytospin 4 centrifuge, by Thermo Fisher Scientific (1 mentions)
Elyra s 1, by Zeiss (1 mentions)
Nuclei pure prep nuclei isolation kit, by Merck Group (1 mentions)
Axio imager m2, by Zeiss (1 mentions)
Plan apochromat 10 0.45 m27, by Zeiss (1 mentions)
Vt1200 vibratome, by Leica (1 mentions)
7 protocols using zen black 2.3 sp1
1
Isolation and Imaging of Neuronal Nuclei
2
Confocal and Wide-field Microscopy Techniques for Tissue Analysis
3
Multimodal Microscopy for Biological Imaging
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All images in this study were acquired either with a Zeiss LSM 880 confocal microscope using the objectives Fluar 5× 0.25 M27, Plan-Apochromat 10× 0.45 M27 (working distance 2.0 mm), and Plan-Apochromat 25× 0.8 Imm Corr DIC M27 multi-immersion; or with a Keyence BZ-X700 microscope (see Supplementary Note 24 ). The acquired images were processed in the respective microscope softwares Zen Black 2.3 SP1 (Zeiss), BZ-X Analyzer (Keyence), Keyence Hybrid Cell Count software (BZ-H3C), ImageJ, Imaris (Bitplane) and with Photoshop CC 2018 (Adobe). The images were compiled in Illustrator CC 2018 (Adobe).
4
Confocal Microscopy of Airyscan Fast Imaging
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All images were collected with a Zeiss LSM880 confocal microscope equipped with a 10X0.45 NA plan apochromat objective at ×2 magnification, as standard for AiryscanFast collection. All tiled Z-stacks were acquired in AiryscanFast mode, the acquisition method set to Z-stack (per channel), and with a 2.0 µm step size. Scans were conducted using 405, 488, 561, and 633 nm laser lines, and emissions were collected using standard Airyscan filters and the Airyscan detector, in Zen Black 2.3 SP1 (Carl Zeiss, RRID:SCR_013672 ). Final voxel sizes were 0.22 × 0.22 × 1.98 µm3, and no digital gain or detector offset was used. Detector gains and laser intensities were consistent across all samples.
5
Multimodal Microscopy for Biological Imaging
6
AAV Neuroanatomy Mapping in Mice
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AAV vectors were administered intravenously to adult mice via retro-orbital injection at doses of 1 × 1011 or 3 × 1011 v.g. as indicated in figures and legends. After 3 weeks of expression, mice were anesthetized with Euthasol (pentobarbital sodium and phenytoin sodium solution, Virbac AH) and transcardially perfused with roughly 50 ml of 0.1 M PBS (pH 7.4) and then another 50 ml of 4% paraformaldehyde (PFA) in 0.1 M PBS. Organs were then harvested and postfixed in 4% PFA overnight at 4°C before being washed and stored in 0.1 M PBS and 0.05% sodium azide at 4°C. Last, the brain was cut into 100-μm sections on a Leica VT1200 vibratome. Images were acquired with a Zeiss LSM 880 confocal microscope using a Plan-Apochromat 10× 0.45 M27 (working distance, 2.0 mm) objective and processed in ZEN Black 2.3 SP1 (Zeiss) and ImageJ software.
7
Lens Microstructural Analysis Protocol
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Raw fluorescent images were processed using Zen Black 2.3SP1 (Zeiss) software. FIJI software was used for lens morphometric analysis and measurements of microstructural features as previously described (Parreno et al., 2018) . Capsule thickness was measured by obtaining intensity distributions of capsular (WGA-640) and basal epithelial F-actin (rhodaminephalloidin) stains using line scan analysis of XZ plane-view reconstructions and by performing subtractive peak-to-peak analysis of fluorescent pixel intensity to obtain distance (Parreno et al., 2018) .
Epithelial cell area was calculated by tracing a population of at least 30 cells (region of interest; ROI) whose boundaries were identified by staining of F-actin, using rhodaminephalloidin, at cell membranes. The total number of cells within the ROI was determined by counting cell nuclei that were stained with Hoechst. Average cell number was calculated using the equation, average cell area = ROI area / total number of cells.
Fiber cell width was calculated by line scan analysis of fiber cell membranes stained with rhodamine-phalloidin at the lens equator, ~10μm inward from the fulcrum. On FIJI, the Distributed Deconvolution (Ddecon) plugin with Z-line predictive model was used to provide high spatial precision when analyzing fiber cell widths.
Epithelial cell area was calculated by tracing a population of at least 30 cells (region of interest; ROI) whose boundaries were identified by staining of F-actin, using rhodaminephalloidin, at cell membranes. The total number of cells within the ROI was determined by counting cell nuclei that were stained with Hoechst. Average cell number was calculated using the equation, average cell area = ROI area / total number of cells.
Fiber cell width was calculated by line scan analysis of fiber cell membranes stained with rhodamine-phalloidin at the lens equator, ~10μm inward from the fulcrum. On FIJI, the Distributed Deconvolution (Ddecon) plugin with Z-line predictive model was used to provide high spatial precision when analyzing fiber cell widths.
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