Imaging was performed using a Leica SP8 Inverted Tandem Head confocal microscope with LAS X v.3.5.1.18803 software (University of Manchester Bioimaging facility), using 20×, 40×, and 100× magnifications. Deconvolution was performed using Huygens Pro v16.05 software. Protein fluorescence levels were measured using FIJI for Macintosh. From each picture, five measurements of background mean intensity were taken. Each single-cell measurement was then adjusted using the formula: integrated density of nucleus – (area of nucleus × background mean).
Las x v 3
Manufactured by Leica
LAS X v.3.5.1.18803 is a software application developed by Leica for the control and analysis of microscope images. The software provides a platform for image acquisition, processing, and management.
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Lab products found in correlation
Sp8 inverted tandem head confocal microscope, by Leica (2 mentions)
Lsm 700, by Zeiss (2 mentions)
Bz x800 microscope, by Keyence (1 mentions)
Sp8 confocal microscope, by Leica (1 mentions)
Plan apochromat oil objective, by Zeiss (1 mentions)
Hc pl apo cs2 63 1.40 oil, by Leica (1 mentions)
Plan apochromat oil objective, by Leica (1 mentions)
Las x software, by Leica (1 mentions)
Tcs sp8 inverted microscope, by Leica (1 mentions)
Lightning mode, by Leica (1 mentions)
Dmi6000, by Leica (1 mentions)
4 protocols using las x v 3
1
Confocal Microscopy Protein Quantification
2
Confocal Microscopy and Membrane Segmentation
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Imaging was performed using a Leica SP8 Inverted Tandem Head confocal microscope with LAS X v.3.5.1.18803 software (University of Manchester Bioimaging facility), using 40×, and 100× magnifications. Deconvolution was performed using Huygens Pro v16.05 software. Membrane segmentation was performed on Imaris (version 9.5.0), mRNA molecules and Transcription sites were counted after membrane segmentation on Imaris 9.5.0 using the Cell module. Protein fluorescence levels were measured using FIJI for Macintosh. From each picture, five measurements of background mean intensity were taken. Each single measurement was then adjusted using the formula: integrated density—(area × background mean).
3
Quantitative Analysis of Brain Engraftment
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We acquired epifluorescence images using a BZ-X800 Microscope (Keyence). Whole brain tiled images were taken at 10× magnification and stitched using Keyence Analyzer software prior to image export. Representative images were taken at 10×. We analyzed images in Fiji (https://imagej.net/Fiji ), adjusting for brightness and black values, but we performed no other image math. Engraftment renderings were made in Fiji (example, Fig. S1 A ). The 488/GFP channel was processed by subtracting background, adjusting threshold level, analyzing particles (size = 0–0.1 in2; circularity = 0.5–1), making a mask of GFP+ cells similar to masking strategy used by Kozlowski et al. (Kozlowski and Weimer, 2012 (link)), converting into ultimate points, and overlaying onto the DAPI channel. The entire brain was then outlined using ImageJ’s “polygon sections” tool and anything falling outside of this brain outline was excluded (example, Fig. S1 J ). Representative confocal images were captured using a Leica SP8 confocal microscope equipped with a Diode 405 laser, HyD sensors, 40×/1.30 oil lens, and controlled with Leica LAS X v.3.5.2 software. Z-stack processed using maximum intensity projection function in ImageJ.
4
Ratiometric Imaging of Mitochondrial Redox
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Ratiometric imaging of mito-GRX1-roGFP was performed on an inverted microscope (DMI 6000; Leica) equipped with hybrid photon counting detectors (HyD; Leica). The sample was excited sequentially frame by frame at 408 nm and 488 nm with the detection set to 500-535 nm. Fluorescence was collected through a 63× 1.40 NA oil immersion objective (HC PL APO 63×/1.40 Oil CS2; Leica). Images were captured using the LAS X software (Leica). All imaging was performed at 37 °C in pre-warmed Leibovitz medium for maximum 90 min per sample.
For confocal microscopy of fixed BrU samples the imaging was performed using a Leica TCS SP8 inverted microscope equipped with 405-, 488-, 552-and 638-nm lasers and a Plan-Apochromat oil objective (×63, NA 1.4). The Lightning mode (Leica) was used to generate deconvolved images. Microscope acquisitions were controlled by LAS X (v. 3.5.2) software from Leica.
CLEM and caspase 3/caspase 7 samples were imaged on a Zeiss LSM 700 inverted confocal microscope equipped with a Plan-Apochromat oil objective (×63, NA 1.40) and 488-nm and 555-nm solid-state lasers and three photomultipliers. Acquisitions were controlled by the Zeiss Zen (v. 6.0.0) software.
For confocal microscopy of fixed BrU samples the imaging was performed using a Leica TCS SP8 inverted microscope equipped with 405-, 488-, 552-and 638-nm lasers and a Plan-Apochromat oil objective (×63, NA 1.4). The Lightning mode (Leica) was used to generate deconvolved images. Microscope acquisitions were controlled by LAS X (v. 3.5.2) software from Leica.
CLEM and caspase 3/caspase 7 samples were imaged on a Zeiss LSM 700 inverted confocal microscope equipped with a Plan-Apochromat oil objective (×63, NA 1.40) and 488-nm and 555-nm solid-state lasers and three photomultipliers. Acquisitions were controlled by the Zeiss Zen (v. 6.0.0) software.
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