4 × 104 skeletal muscle cells were seeded onto Gibco Geltrex-coated 13 mm round coverslips (VWR, UK) in 24 well plates. After differentiation, cells were fixed with 4% formaldehyde solution for 10 min and permeabilized in 0.1% triton X 100 for 15 min, then blocked with 5% goat serum for up to 1 hr. Primary antibodies were incubated by inverting the coverslip onto a 30 µL drop of antibody solution on Parafilm overnight at 4°C (see Table S1 in the Supplementary Material for comprehensive details of the antibodies and the dilutions used). Alexafluor-conjugated secondary antibodies were added for 1 hr at room temperature. HCS CellMask deep red or red was added at 1 : 5,000 for 30 min followed by 5 µg/mL 4′, 6-diamidino-2-phenylindole (DAPI) counterstaining. Coverslips were mounted onto glass slides with DAKO fluorescence mounting medium (Agilent, USA) and imaged using either an Olympus IX81-ZDC inverted widefield microscope or a Zeiss LSM 980 Airyscan 2 confocal microscope.
Lsm 980 airyscan 2 confocal microscope
Manufactured by Zeiss
Sourced in United States
The ZEISS LSM 980 Airyscan 2 is a confocal microscope that utilizes advanced Airyscan technology to provide high-resolution and high-sensitivity imaging. It features a unique detector design that enables efficient light collection and improved signal-to-noise ratio. The LSM 980 Airyscan 2 is designed for a wide range of applications in life science research.
Automatically generated - may contain errors
Lab products found in correlation
Zen blue software, by Zeiss (2 mentions)
Dako fluorescence mounting medium, by Agilent Technologies (1 mentions)
Cryomatrix, by Thermo Fisher Scientific (1 mentions)
Prolong antifade, by Thermo Fisher Scientific (1 mentions)
Plan apochromat 63 1.4 oil dic objective, by Zeiss (1 mentions)
Axiovision software, by Zeiss (1 mentions)
Prolong gold antifade mountant with dapi, by Thermo Fisher Scientific (1 mentions)
Plan apochromat 63 1.4 oil, by Zeiss (1 mentions)
Ab5535, by Merck Group (1 mentions)
Sp5 upright confocal microscope, by Leica (1 mentions)
Ab13970, by Abcam (1 mentions)
8 protocols using lsm 980 airyscan 2 confocal microscope
1
Immunocytochemical Analysis of Skeletal Muscle Cells
2
Cryosectioning and Immunostaining of Runx2-Cre x TdTomato Mouse Femurs
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Femurs from 6-month-old Runx2-Cre x TdTomato mice were fixed in 4% paraformaldehyde (PFA) at 4 °C for 72 h under gentle agitation. Bones were decalcified in 10% EDTA for 2 weeks at 4 °C under gentle shaking agitation, followed by incubation in 30% sucrose for 3 days at 4 °C. Samples were embedded in Cryomatrix (ThermoFisher Scientific, Wilmington, DE) and flash frozen in liquid nitrogen and stored at −80 °C. 7 μm-thick cryosections were prepared for fluorescent imaging. Bone sections were stained with anti-mouse CD24 antibody (Biolegend) at 1:50 dilution at 4 °C overnight, followed by anti-Rat-FITC secondary antibody at 1:200 for 1 h at room temperature. Slides were then mounted with ProLong Antifade (ThermoFisher) and imaged on an LSM 980 Airyscan 2 confocal microscope (Carl Zeiss Microscopy) at 63X magnification.
3
Visualizing THUMPD3 Protein Localization
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HEK293T cells were transfected with pcDNA3.1(+)-THUMPD3-HA plasmid. After transfection for 24 h, the cells were fixed in 4% paraformaldehyde for 10 min and then permeated in 0.2% Triton X-100 for 5 min on ice. After washing with phosphate-buffered saline (PBS), the fixed cells were blocked in PBS containing 5% BSA and then incubated with rabbit anti-HA antibodies with 1:800 dilution overnight at 4°C. The cells were then immunolabeled with Alexa Fluor 488-conjugated goat anti-rabbit IgG in PBS with 1:100 dilution for 2 h and the nuclear counterstain DAPI for 5 min at room temperature. Fluorescent images were taken and analysed using an LSM980 Airyscan2 confocal microscope (Zeiss).
4
Immunofluorescence Analysis of Exogenous THUMPD2
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HEK293T cells were transfected with pcDNA3.1(+)-THUMPD2-HA plasmid. After transfection for 36 h, the cells were fixed in 4% paraformaldehyde for 10 min and then permeated in 0.2% Triton X-100 for 5 min on ice. After washing with phosphate-buffered saline (PBS), the fixed cells were blocked in PBS containing 5% bovine serum albumin (BSA) and then incubated with mouse anti-HA antibody (6E2) with 1:800 dilution overnight at 4°C. The cells were then immunolabeled with Alexa Fluor 647-conjugated donkey anti-mouse IgG in PBS with 1:400 dilution for 1 h and the nuclear counterstain DAPI for 5 min at room temperature. Fluorescent images were taken and analyzed using a LSM980 Airyscan2 confocal microscope (Zeiss).
5
Fluorescence Imaging of Adherent Cells
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For fluorescence imaging, cells were grown adherently on glass cover slips to 50% confluency. After washing the cells with pre-warmed (37°C) PBS, they were fixed with 3.7% paraformaldehyde in PBS for 10 min at 37°C. The fixation was stopped by replacing the solution with 100 mM glycine in PBS for 5 min at 37°C. After that, the cells were washed twice with PBS, mounted on the specimen slide with the help of a drop of Prolong Gold Antifade Mountant with DAPI (P36941; Thermo Fisher Scientific), and dried in the dark at least overnight.
The fluorescent specimens were imaged using a Plan-Apochromat 63×/1,4 Oil DIC Objective at a Zeiss LSM980/Airyscan 2 confocal microscope. sfGFP was excited by a 488-nm diode laser and emission was detected using a 300–720-nm band pass filter. Separately, DAPI was excited by a 405-nm diode laser and emission was detected using a 300–720-nm band pass filter. For the 3D model, a Z-stack was imaged using the internal GaAsP-PMT detectors from 490 to 668 nm for sfGFP and 410–473 nm for DAPI in a two-track process. Image processing was carried out using the Zeiss AxioVision software. The 3D Volume images were created in Imaris 9.6.
The fluorescent specimens were imaged using a Plan-Apochromat 63×/1,4 Oil DIC Objective at a Zeiss LSM980/Airyscan 2 confocal microscope. sfGFP was excited by a 488-nm diode laser and emission was detected using a 300–720-nm band pass filter. Separately, DAPI was excited by a 405-nm diode laser and emission was detected using a 300–720-nm band pass filter. For the 3D model, a Z-stack was imaged using the internal GaAsP-PMT detectors from 490 to 668 nm for sfGFP and 410–473 nm for DAPI in a two-track process. Image processing was carried out using the Zeiss AxioVision software. The 3D Volume images were created in Imaris 9.6.
6
Live Imaging with Airyscan Confocal Microscopy
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Live imaging experiments were performed using the LSM980 Airyscan 2 confocal microscope (Carl Zeiss Microscopy) equipped with the C-Apochromat 40×/1.20 W Korr or the Plan-Apochromat 63×/1.4 oil differential interference contrast objective.
7
Cell Morphology Imaging in Nematodes
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To image the morphology of cells, mitochondria, and plasma membranes in various cell types, nematodes hosting the yphEx301(Posm-6::MTS::roGFP), yphEx302(Pmyo-2::MTS::roGFP), zcIs14(Pmyo-3::MTS::GFP), yphIs17(Pcol-19::MTS::GCaMP5), yphEx306(Pmyo-3::myrGFP), yphEx307(Pklp-6::myrGFP), yphEx309(Pcol-19::memGFP), and yphEx216(Posm-6::GFP) plasmids were used. Animals were exposed to P. ostreatus or 1 μl of 80% 3-octanone for 5 or 10 min, then placed on 3% agar pads on glass slides with 2 μl of 500 mM NaN3, and imaged using an LSM 980 Airyscan 2 confocal microscope (Carl Zeiss) with a C-Apochromat 63×/1.2 numerical aperture (NA) water immersion objective. Images were acquired with Multiplex Mode (SR-4Y) and analyzed in Zeiss ZEN blue software.
8
Embryonic Tissue Imaging and Characterization
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Embryos were dissected under a Leica fluorescent stereomicroscope and fixed for 1h in 4% formaldehyde at RT. For sections preparations, embryos were embedded in 15% sucrose/7.5% gelatine/PBS solution and sectioned with Leica cryostat at 20µm. Antibody staining was performed as described in (Serralbo and Marcelle, 2014) (link). The following primary antibodies were used: anti-GFP chicken polyclonal (ab13970, Abcam;1/1000), anti-Pax7 IgGI mouse monoclonal (Hybridoma Bank; 1/10), Rabbit polyclonal anti-Sox9 (ab5535, Millipore; 1/1000), anti-Myosin heavy Chain IgGIIb (Hybridoma bank; 1/10), anti-HNK1 IgGM (Hybridoma bank; 1/10). Images of stained sections were acquired with a Leica SP5 upright confocal microscope and an UV-corrected HCX PL APO CS 40x / NA 1.25 Oil immersion objective (WD 0.1 mm), combined with tile scan acquisition. For whole-mount samples, a CARL ZEISS LSM 980 Airyscan 2 confocal microscope on inverted Axio Observer stand was used, with UV-IR corrected PL APO 40x/1.3 oil immersion objective (WD 0.20 mm).
Images of native reporter activity in E9 embryos were taken under a Leica 3D Fluorescent microscope 4x dry objective.
Images of native reporter activity in E9 embryos were taken under a Leica 3D Fluorescent microscope 4x dry objective.
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