The scaffolds were rinsed with PBS three times and then methanol was added in order to fix the adhered cells. They were kept for 20 min at room temperature; thereafter, they were stored at −20 °C. The scaffolds were rinsed three times with PBS. The samples were then incubated at room temperature and in dark with DiOC6(3) (D273, Life Technologies, Eugene, Oregon) diluted at 1:700 in PBS. After 45 min, the liquid was aspirated and propidium iodide (P4864, Sigma Aldrich, Darmstadt, Germany) in dilution 1:200 in PBS was added for 10 min. Finally, the samples were washed with PBS three times. For scanning, we used a confocal microscope Zeiss LSM 510 DUO (Zeiss, Jena, Germany). Cell nuclei stained with propidium iodide are visualized in red, cytoplasmic membranes in green color. The experiment was carried out in three biological repeats per group. Representative images are presented.
Lsm 510 duo
Manufactured by Zeiss
Sourced in Germany
The LSM 510 DUO is a multi-photon microscope system developed by Zeiss. It is designed for advanced imaging applications that require high-resolution, deep tissue penetration, and fast acquisition speeds. The system combines two laser sources, enabling both confocal and multi-photon imaging capabilities in a single platform.
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Lab products found in correlation
Lci plan neofluar 63 1.3 water immersion dic m27 objective, by Zeiss (2 mentions)
Dioc6 3, by Thermo Fisher Scientific (1 mentions)
P4864, by Merck Group (1 mentions)
Atropine sulfate, by Merck Group (1 mentions)
Zen 2009 imaging software, by Zeiss (1 mentions)
Plan apochromat 40 1.4 oil dic objective, by Zeiss (1 mentions)
Turbofect reagent, by Thermo Fisher Scientific (1 mentions)
Zen 2010, by Zeiss (1 mentions)
Poly l lysine (pll), by Merck Group (1 mentions)
Turbofect, by Thermo Fisher Scientific (1 mentions)
Mitotracker deep red fm, by Thermo Fisher Scientific (1 mentions)
Axiovert 200, by Zeiss (1 mentions)
Fluoromount g, by Southern Biotech (1 mentions)
Vectashield, by Vector Laboratories (1 mentions)
Uas mcd8 gfp, by Bloomington Drosophila Stock Center (1 mentions)
Lsm 510 meta, by Zeiss (1 mentions)
Fluo 4 am, by Thermo Fisher Scientific (1 mentions)
Draq5, by Cell Signaling Technology (1 mentions)
Alexa fluor 488 antibody, by Thermo Fisher Scientific (1 mentions)
Anti acetylated tubulin antibody, by Merck Group (1 mentions)
16 protocols using lsm 510 duo
1
Cell Morphology Visualization on Scaffolds
2
Rat Optic Nerve Vascular Imaging
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Rodent ON vascular filling was performed following Nicholson et al (2012) [16 (link)]. Quantitative ON vascular analysis was performed using tissue from terminally anesthetized rats. After euthanasia, animals were placed on a warming pad (~38°C) and transcardially perfused sequentially with the following heated (~38°C) solutions: 120 ml heparinized saline (50 units/ml) with 2 μg/ml atropine sulfate (Sigma Chemicals) and 100 μM adenosine (HAAS solution), 50 ml fluorescein-conjugated bovine serum albumen (FITC-BSA) with 2% dissolved gelatin (300 bloom, Sigma Chemicals) in HAAS solution, 20 ml FITC-BSA with 4% dissolved gelatin in HAAS solution. Tissues were fixed in 4% neutral-buffered paraformaldehyde (PFA), washed in phosphate buffered saline (PBS), embedded in 10% gelatin (#G1890, Sigma), and re-fixed for an additional 24 h in PFA. Tissues were cryosectioned to 40 μm and imaged by confocal microscopy using tiled z-stacks imaged on a Zeiss LSM510 Duo (Carl Zeiss Microscopy, Gottingen) fitted with a 40x 1.4 NA oil objective. Vascular data was quantified using a filament model constructed using Imaris software (Bitplane Software) (See Nicholson et al. (2012) for method details [16 (link)]).
3
Fluorescent Microscopy of Basidioascus Meiosis
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To study nuclear behaviour and to look for indicators of meiosis, Basidioascus undulatus DAOM 241956 was grown on corn meal agar (CMA, Acumedia Manufacturers, Lansing, MI) for 1 wk and mounted in DNA stains: DAPI-Fluoromount-GTM mounting medium (EMS, Hatfield, PA) or aqueous SYTO 9 (25 μM) (Life Technologies, Burlington, ON). Samples were visualized under confocal laser scanning microscopy using an LSM 510 DUO (Carl Zeiss MicroImaging, Göttingen, Germany) with a Plan-Apochromat 40×/1.4 Oil DIC objective and electronic zoom 4. An excitation diode laser (405 nm) and emission light (420–700 nm) were used for DAPI. An excitation Argon laser (488 nm) and emission light (505–550 nm) were used for SYTO 9. Images were captured using ZEN 2009 Imaging Software (Carl Zeiss MicroImaging).
4
FLIP Imaging of Mitochondrial Proteins
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HCT116 Bax/Bak DKO cells were seeded on a Lab-TekTM chambered coverglass system (VWR) and incubated for 24 to 48 h in McCoy’s 5A medium. Cells were transfected with GFP-tagged constructs containing the HK1, HK2, BAX, BAK, tBid, Bim, or Puma coding sequence (CDS) using Turbofect reagent (Thermo Fisher Scientific) according to manufacturer’s instructions.
FLIP experiments were performed as described previously (12 (link)). For each measurement, one cell expressing GFP-tagged protein in the neighborhood of at least one labeled cell was selected. Cells were imaged prior to bleaching using a Zeiss LSM 510 DUO inverted, high-speed confocal microscope equipped with a three-channel and Meta detector and an LCI-Plan Neofluor 63× lens. A single spot in the analyzed cell was repeatedly bleached with 20 iterations using a 488-nm laser line (75 to 100% output). After each bleaching cycle, two pictures were collected. One FLIP experiment was completed after 10 cycles of repeated bleaching and image acquisition. The Zeiss ZEN 2010 software was used to analyze the fluorescence loss on the mitochondria of the selected cell. Additionally, an unbleached control cell was monitored during the measurement and used to exclude any photobleaching events that may occur during image acquisition.
FLIP experiments were performed as described previously (12 (link)). For each measurement, one cell expressing GFP-tagged protein in the neighborhood of at least one labeled cell was selected. Cells were imaged prior to bleaching using a Zeiss LSM 510 DUO inverted, high-speed confocal microscope equipped with a three-channel and Meta detector and an LCI-Plan Neofluor 63× lens. A single spot in the analyzed cell was repeatedly bleached with 20 iterations using a 488-nm laser line (75 to 100% output). After each bleaching cycle, two pictures were collected. One FLIP experiment was completed after 10 cycles of repeated bleaching and image acquisition. The Zeiss ZEN 2010 software was used to analyze the fluorescence loss on the mitochondria of the selected cell. Additionally, an unbleached control cell was monitored during the measurement and used to exclude any photobleaching events that may occur during image acquisition.
5
Visualizing Chemokine Signaling in Zebrafish
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Zebrafish embryos carrying the hsp70:cxcl12a transgene were heat-shocked for 45 min in a 38°C water bath. The heat-shocked embryos were embedded in 1% low-melting agarose for imaging. Time-lapse imaging was carried out on a ZEISS LSM 510 DUO with a 63×/1.2NA objectives. The embryos injected with the cmtm4 and control MOs were simultaneously heat-shocked and imaged using multiwell dishes.
6
CXCR4b Expression Imaging Workflow
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Lifetime analysis was carried out as previously described (Dona et al., 2013 (link)). Z stacks for cxcr4b:tFT imaging were obtained with a ZEISS LSM 510 DUO using a 63×/1.3NA objective and 488 and 561 nm laser lines.
7
Mitochondrial Imaging in HCT116 Cells
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HCT116 wild-type cells were seeded on coverslips coated with Poly-L-lysine (Sigma-Aldrich) in McCoy’s 5A medium. After 48 h incubation, cells were transfected using Turbofect (Thermo Fisher Scientific) according to manufacturer’s instructions. Then, cells were incubated with MitoTracker Deep Red FM (Thermo Fisher Scientific) for 10 min at 37 °C and fixed with 4% paraformaldehyde (PFA) for 10 min at room temperature. Finally, samples were mounted in Vectashield mounting medium (Linaris) and imaged using a ZEISS LSM 510 DUO with inverted microscope Axiovert 200 equipped with 488 and 633 nm lasers lines and a 63× PlanFluor lens.
8
Multimodal Tissue Imaging Protocol
9
Mitochondrial Fusion Rate Analysis
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For mitochondrial fusion rate analysis, cortical neuronal cultures transfected with mito-KikGR1 plasmid and plasmids of interest as described earlier [39 (link)] and examined at DIV 7–8. A laser scanning confocal microscope (LSM 510 Duo, Carl Zeiss Microscopy GmbH) equipped with a LCI Plan-Neofluar 63×/1.3 water immersion DIC M27 objective was used. The temperature was maintained at 37°C using a climate chamber. For fusion acquisition, mito-KikGR1 was illuminated with a 488-nm argon laser line to visualize the intense green mitochondrial staining. Selected mitochondria were then photoconverted to red using a 405-nm diode laser and illuminated using a 561 nm DPSS laser. The images were taken at 10-s intervals for 10 min, the fate of all activated mitochondria was followed throughout the time-lapse, and the fusion and fission events were recorded.
10
Visualizing R14H06-GAL4 Expression in Adult Brains
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To observe the expression domain of R14H06-GAL4 in adult brains we crossed this line to UAS-mCD8-GFP (Bloomington stock # 5137) and UAS-unc84::GFP and performed confocal microscopy. Brains were dissected in PBS and fixed with 4% paraformaldehyde for 45 min at room temperature. Counterstaining was performed with nc82 primary antibodies (1:50 dilution – developmental studies hybridoma bank) and DyLight 594 secondary antibodies (1:400 dilution). Brains were mounted in Vectashield (Vector Laboratories) and imaged using a Zeiss LSM 510 duo vario confocal microscope. Confocal projections were captured with 1 μm slices and processed using Image J software (Fiji) and Adobe Photoshop (Schindelin et al. 2012 (link)).
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