Cells were quickly rinsed with PBS and fixed with 4% paraformaldehyde at room temperature for 15 min. Paraformaldehyde was then neutralized with NH4Cl for 15 min before cells were permeabilized with 0.5% Triton X-100 for 5 min, washed three times with PBS, rinsed with PBS 5% BSA for 40 min, then incubated overnight at 4°C with primary antibodies, followed by washes and incubation with Alexa Fluor® 633 goat anti-rabbit IgG (H + L) (Molecular Probes, A21071; 1:1,000) for 1 h at room temperature (Xiao et al., 2019 (link)). Next, cells were rinsed with PBS, incubated with 5 μg/mL Hoechst 33342 (Sigma) for 5 min, washed again with PBS, and mounted with 15 μL Mowiol® 4–88 (Calbiochem, San Diego, CA, United States). Cells were examined with a confocal microscope (Leica TCS-SP8 gated STED) (Yan et al., 2018 (link)). Alexa Fluor 633 was excited at 633 nm with a white light laser, and emission measured at 640–800 nm with a hybrid detector. Hoechst 33342 was excited by a 405-nm diode, and emission measured at 420–460 nm.
Tcs sp8 gated sted
Manufactured by Leica
Sourced in Germany
The TCS SP8 gated STED is a high-performance confocal imaging system designed for advanced fluorescence microscopy applications. It combines confocal laser scanning microscopy with stimulated emission depletion (STED) technology to achieve super-resolution imaging beyond the diffraction limit.
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5 protocols using tcs sp8 gated sted
1
Immunofluorescence staining of cells
2
Super-Resolution Microscopy of Nematodes
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Super‐resolution microscopy was performed using the Leica TCS SP8 gated STED. This instrument is an inverted microscope (Leica DMI6000 CS) equipped with a 100× oil objective lens and a White Light Laser (WLL). Images were taken with a resolution of about 60 nm (pixel size of 18.93 × 18.93 nm) following the use of a depletion laser at 592 nm. In all the experiments, nematodes were paralyzed with 25 mM Levamisole (AppliChem) in M9 buffer mounted on 2% agarose pads on glass slides and closed with coverslips. FRAP experiments were conducted blind to the respective condition (genotype, treatment, or RNAi) of the nematodes.
3
Multimodal Cardiac Imaging with gSTED
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Imaging of heart sections was performed using a Leica TCS SP8 gated STED (gSTED) microscope, equipped with a white light laser and a 93x objective lens (HC PL APO CS2 93x GLYC, NA 1.30). For confocal images of mitochondria and DNA, Z-stacks in accordance with the Nyquist sampling criteria were taken by exciting the fluorophores at 488 nm and 594 nm, respectively, and Hybrid detectors collected fluorescent signals. Stimulated emission depletion of DNA channel was performed with a 775 nm depletion laser. 2D confocal and gSTED images were acquired sequentially with the optical zoom set to obtain a voxel size of 17 x 17 nm. Excitation was provided at 594 nm and Hybrid detectors collected signal. Gating between 0.3–6 ns was applied. Images were deconvolved with the Huygens software. Performance of the microscope and optimal depletion laser power were tested as previously described [65 (link)].
4
STED Imaging of Mitochondria and DNA
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Imaging was performed with a Leica TCS SP8 gated STED (gSTED) microscope, with a white light laser and a 93× objective lens (HC PL APO CS2 93× GLYC, NA 1.30). For confocal images of mitochondria (TOM20) and DNA, Z-stacks were taken by exciting the fluorophores at 488 and 594 nm, respectively, and Hybrid detectors collected fluorescent signals. STED images of DNA channel were obtained with a 775-nm depletion laser. 2D confocal and gSTED images were acquired sequentially with the optical zoom set to obtain a voxel size of 17 × 17 nm. Excitation was provided at 594 nm and Hybrid detectors collected signal. Gating between 0.3 and 6 ns was applied. Performance of the microscope and optimal depletion laser power were tested as previously described (Nicholls et al, 2018 (link)).
5
Confocal Microscopy of Emulsion Droplets
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The microscopic structure of the emulsions was observed using a confocal scanning laser microscope (inverted Leica TCS SP8-gated STED, Germany) equipped with a WLL laser (488 and 563 nm excitation wavelengths) using a HC PL APO CS2 63x/1.40 oil immersion objective lens. 20 µL were taken from the emulsion layer for these experiments. To prevent the deformation of emulsion droplets, the sample was placed in a curved glass slip with a cover glass slide on the top. The NPs were labeled in red with rhodamine as previously described (see section 2.2). The aqueous phase was labeled in green by re-suspension of the lyophilized PLGA-PVA NPs in a 0.08 mg/mL solution of Oregon green instead of MilliQ water or by the addition of 100 µL of calcein solution at 0.4 mg/mL to the PLGA NP suspension. Red fluorescence was observed with a 600-710 nm filter under a 563 nm laser illumination. Green fluorescence was observed with a 500-535 nm filter under a 488 nm laser illumination. A hybrid detector under a gated mode (0.3-6.5 ns) was used in order to avoid reflection. The green and the red fluorescence emissions were collected under a sequential mode. The pinhole was set at 1.0 Airy unit.
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