For immunofluorescence analysis, RAW264.7 cells were seeded on glass coverslip in a six-well plate at a density of 2 × 105 cells/well, and then incubated overnight. After being treated with 10 μM DC or SFN for 6 h, the cells were fixed with 4% paraformaldehyde for 30 min at room temperature, and subsequently permeabilized with 0.1% Triton X-100 for 30 min and blocked with 5% BSA for 30 min. The cells were then incubated with Nrf2 antibody for overnight, next incubated with the secondary antibody (Alexa Fluor 488-conjugated secondary antibody) for 1 h. At last, the cells were stained with DAPI for 5 min. The fluorescence images were captured by using a Leica TCS SP8 Confocal Laser Scanning Microscope System (Leica, Wetzlar, Germany).
Tcs sp8 confocal laser scanning microscope system
Manufactured by Leica
Sourced in Germany, United States
The TCS SP8 is a confocal laser scanning microscope system manufactured by Leica. It is designed to provide high-resolution imaging and analysis of samples. The system utilizes a combination of laser illumination, confocal optics, and sensitive detectors to capture detailed images of biological and material samples.
Automatically generated - may contain errors
Lab products found in correlation
Cm 1000 cryostat, by Leica (2 mentions)
Dapi solution, by Thermo Fisher Scientific (1 mentions)
Confocal dishes, by NEST Biotechnology (1 mentions)
Las x software, by Leica (1 mentions)
Mito tracker green, by Beyotime (1 mentions)
Lyso tracker red, by Beyotime (1 mentions)
Alexa fluor 488 conjugated goat anti mouse igg, by Cell Signaling Technology (1 mentions)
Ab683 clone 1g12, by Abcam (1 mentions)
13 protocols using tcs sp8 confocal laser scanning microscope system
1
Nrf2 Activation by DC and SFN
2
Immunofluorescence Staining of NF-kB in BV-2 Microglia
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For BV-2 microglia cells staining, cells were washed, fixed by 3.7% PFA, permeabilized by 0.3% Triton X-100-PBS for 15 min, and then blocked for another 30 min at room temperature. After that, cells were incubated with rabbit anti-NF-kB p65 (1:500) overnight at 4°C and goat anti-rabbit, Alex Fluor 594 conjugate secondary antibody (111-585-003, 1:500, Jackson Immuno Research Laboratories, West Grove, PA, United States) for 1 h at room temperature in the dark. For the nuclei observation, the cells were further stained with DAPI solution (Thermo Scientific) for 10 min. Finally, the samples were mounted with the Prolong anti-fade reagent and imaged by the Leica TCS SP8 confocal laser scanning microscope system (Leica, Wetzlar, Germany).
3
Flavonol Modulation of NF-κB Activation
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RAW 264.7 cells (1 × 104) were cultured in the confocal dishes (NEST Biotechnology Co., Ltd, China) overnight. Then, cells were pretreated with the highest concentration of selected flavonols for 4 h, followed by inducing LPS (1 μg/mL) for 30 min. After treatment and stimulation, cells were washed with cold PBS for 3 times and immediately fixed in 4% paraformaldehyde for 15 min. Cold PBS was used to washed the cells for another three times and 0.5% Triton X-100 solution was added for cell permeabilization for 20 min at room temperature. Next, cells were blocked with 3% BSA for 1 h in the cell incubator and followed by incubating with primary antibody rabbit NF-κB p65 (1: 500 dilution) for 2 h at room temperature. Cells were washed with ice-cold PBS for 3 times and incubated with secondary antibody Alexa Fluor 568 goat anti-rabbit IgG (H + L) (1:500 dilution) for 1 h at dark at room temperature. Lastly, cells were stained by 4′,6-diamidino-2-phenylindole (DAPI) for 10 min at dark for labeling cell nucleus and photographed by Leica TCS SP8 Confocal Laser Scanning Microscope System (Leica, USA).
4
Quantitative Imaging of Neuromuscular Junctions
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Z‐stack images of whole‐mount immunofluorescence stained EDL, 40‐µm‐thick longitudinal TA, and soleus sections, and cross‐sections of human gluteus medius were acquired using the TCS SP8 confocal laser scanning microscope system (Leica), and reconstructed images were visualized by maximum intensity projection using LAS X software (Leica). For denervation assessment, we analyzed in 50–100 NMJs of EDL muscle per mouse, 20–60 NMJs of TA muscle per mouse, 10–50 NMJs of soleus muscle per mouse. Numbers of completely or partially denervated, and innervated NMJs were counted. For quantification of MFG‐E8 signal intensity, we analyzed in 20–80 NMJs of TA muscle per mouse, 10–50 NMJs of soleus muscle per mouse, 6–17 arteries of gluteus medius per human. The signal intensity of MFG‐E8 was quantified using ImageJ software (NIH). The region of interest (ROI) was manually set to NMJs and arteries, and then, the intensity of the fluorescence signal of MFG‐E8 was measured.
5
Mitochondrial and Lysosomal Dynamics in C2C12 Myotubes
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C2C12 cells were seeded with 1.0 × 105 cells per well in six‐well plates. After fully differentiation, myotubes were treated with 10 μM DEX, with or without different concentrations of MY, for 24 h. Living cells were directly stained with MitoTracker Green or LysoTracker Red probes (Beyotime Biotechnology) following the manufacturer's instructions. Fluorescent images were captured with a Leica TCS SP8 Confocal Laser Scanning Microscope System (Buffalo Grove, IL).
6
Cellular FRET Imaging Protocol
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All cellular fluorescence images were collected with a Leica TCS SP8 confocal laser scanning microscope system. An external excitation of 488 nm was chosen for the green channel of the fluorophore (FAM) donor. The FRET excitation of 488 nm was chosen for the yellow channel of the fluorophore (TAMRA) acceptor. A stimulation of 561 nm was selected for the red channel of the TAMRA fluorophore. All images were developed using a 60× objective lens with oil. The FRET signal was denoted as the fluorescence intensity ratio of FRET/FAM in intracellular imaging systems upon an excitation at 488 nm. The background FRET signal, generated from solely the FAM or TAMRA fluorophore, was subtracted from each of the samples to improve the reliability of our FRET transductions. After incubating the updated CHA–HCR mixture with MCF-7 or Hela cells for 5 h at 37 °C, the Z-stack projections were taken with 51 slice stacks at 0.2 μm increments throughout the entire cells. All FRET images of living cells were normalized by using ImageJ and Fiji software.
7
Cellular Uptake Imaging of Complexes
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A549 cells were seeded into a glass-bottomed dish (35 mm dish with 20 mm well). After 24 h, the cells were incubated with complexes for indicated time periods or concentrations and then washed with phosphate-buffered saline three times. The luminescence imaging of complexes in cells was carried out by a Leica TCS SP8 confocal laser scanning microscope system. The excitation wavelength was 488 nm.
8
Intracellular Trafficking of Nanoparticles
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To investigate the intracellular trafficking of OVA@acGM-8k NPs, DC2.4 cells were incubated with OVA, OVA@acGM-8k NPs and OVA@acDEX NPs. After 2 h incubation, the cellular uptake was determined by flow cytometry. For confocal laser imaging, the DC2.4 was replaced with fresh medium and incubated for another 1, 3, 6 h. Then the DC2.4 cells were stained with Hoechst 33,342 for 15 min and lysotracker red for 15 min and visualized in Leica TCS SP8 Confocal Laser Scanning Microscope System.
9
Luminescence Imaging of Complex 6 in HUVEC Cells
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HUVEC cells were seeded into a glass-bottomed dish (35 mm dish with 20 mm wells) and incubated for 24 h. Complex 6 was added to cells for indicated time periods or concentrations, followed by washing with phosphate-buffered saline (PBS) three times. The luminescence imaging of complex 6 in cells was carried out using a Leica TCS SP8 confocal laser scanning microscope system. The excitation wavelength was 488 nm.
10
Vascular Oxidative Stress Imaging
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Segments of or aortas (chronic treatment) and carotid arteries (ex vivo treatment) were frozen in OCT compound (Tissue-Tek) and sliced into sections (10 μm) using a Leica CM 1000 cryostat. The arterial sections and treated HUVECs were incubated in DHE- (5 μM; Invitrogen) containing normal physiological saline solution (NPSS) at 37°C for 15 min. NPSS contained (mM): 140 NaCl, 5 KCl, 1 CaCl2, 1 MgCl2, 10 glucose, and 5 HEPES (pH 7.4). Fluorescence images were obtained with the Leica TCS SP8 Confocal Laser Scanning Microscope System (Leica Microsystems, Germany) by measuring the fluorescence intensity at excitation 515 nm and emission 585 nm.
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