PK-15 cells grown to approximately 80% confluence in a petri dish with a glass bottom (NEST, GBD-35-20) were infected with FMDV at 1 MOI. After 3 hpi, cells were fixed using methanol for 15 min at -20°C. Cells were blocked with phosphate-buffered saline containing 5% goat serum (Life Technologies, 16210064) for 1 h at room temperature. Next, cells were incubated overnight at 4°C in the presence of primary antibody (anti-LC3B and anti-3D) (1:200), followed by incubation in fluorochrome-conjugated secondary antibody (Alexa Fluor® 488 and 546, 1:200; Thermo, A11070 and A11018) diluted in antibody dilution buffer for 1 h at room temperature in the dark. Then cells were incubated with DAPI (Roche, 10236276001) for nuclear staining. The fluorescence signals were visualized with a TCS SP8 confocal fluorescence microscope (Leica).
Tcs sp8 confocal fluorescence microscope
Manufactured by Leica camera
Sourced in Germany
The Leica TCS SP8 is a confocal fluorescence microscope designed for high-resolution imaging. It features a modular architecture, allowing for customization to suit various research applications. The TCS SP8 utilizes laser excitation and advanced optics to capture detailed fluorescence images with minimal background noise.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (4 mentions)
Goat serum, by Thermo Fisher Scientific (1 mentions)
Gbd 35 20, by NEST Biotechnology (1 mentions)
Alexa fluor 488 and 546, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Roche (1 mentions)
Tcs sp8, by Leica camera (1 mentions)
Renilla luciferase assay system, by Promega (1 mentions)
Glass bottom microwell dishes, by MatTek (1 mentions)
Prolong diamond antifade mountant, by Thermo Fisher Scientific (1 mentions)
Application suite x software, by Leica camera (1 mentions)
Alexa fluor 568, by Abcam (1 mentions)
R37814, by Thermo Fisher Scientific (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Dapi solution, by Merck Group (1 mentions)
Alexa fluor 488 goat anti rabbit igg h l, by Thermo Fisher Scientific (1 mentions)
α actinin, by Merck Group (1 mentions)
Tnnt2, by Merck Group (1 mentions)
Tnni3, by Santa Cruz Biotechnology (1 mentions)
Alexa conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Goat serum, by Merck Group (1 mentions)
22 protocols using tcs sp8 confocal fluorescence microscope
1
Immunofluorescence Analysis of FMDV Infection
2
Evaluating Au4-IO NP-induced Radiosensitization
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4T1 cells were cultured on glass-bottomed petri dishes and treated with or without Au4-IO NP/Au4-IO NP-cRGD (2 μM). After 6 h, the medium was removed, and then the prepared test solution (OH580 Stain Working solution) was added and incubated for 1 h at 37 °C. Then these groups were treated with or without X-ray (4 Gy). Finally, the cells were stained with DAPI, and imaged using Leica TCS SP8 confocal fluorescence microscope at excitation wavelengths of 405 and 540 nm.
3
Transient Expression and Cell Viability Assays in Rice Protoplasts
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Transient expression in rice protoplasts was implemented as previous described (Zhang et al., 2011 (link)). The cell death assays were conducted using rice protoplasts as described (Zhao et al., 2016 (link)). For the cell viability assay, protoplasts were transfected with the indicated plasmids for 20 h and stained with 220 μg/mL fluorescein diacetate (FDA). Each protoplast sample was scored under fluorescence microscope (TCS SP8, Lecia) in at least 10 randomly selected microscopic fields. For the luciferase assay, The Renilla luciferase gene was used as a reporter to monitor protoplast viability. The indicated genes and LUC gene were co-transformed in rice protoplasts with the same quantity level of cells, respectively. Luciferase activity was measured 40 h following transformation using a Renilla luciferase assay system (Promega).
For subcellular localization, Protoplasts preparation were the same as described above. NlSP1-RFP recombinant vector was co-transformed with nuclear-GFP maker bZIP63 (Walter et al., 2004 (link)) and others organelles makers (Nelson et al., 2007 (link)) in rice protoplasts, respectively. After transfection, the protoplasts were placed in a 28°C dark incubator and cultured for 16 to 22 h. The fluorescence of protoplasts was observed and photographed using Leica TCS SP8 confocal fluorescence microscope.
For subcellular localization, Protoplasts preparation were the same as described above. NlSP1-RFP recombinant vector was co-transformed with nuclear-GFP maker bZIP63 (Walter et al., 2004 (link)) and others organelles makers (Nelson et al., 2007 (link)) in rice protoplasts, respectively. After transfection, the protoplasts were placed in a 28°C dark incubator and cultured for 16 to 22 h. The fluorescence of protoplasts was observed and photographed using Leica TCS SP8 confocal fluorescence microscope.
4
Imaging Peptide Uptake in U87MG Cells
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U87MG cells were grown and maintained in DMEM media with 10% FBS and 1% penicillin/streptomycin at 37 °C, 5% CO2. Before staining experiments with peptides, the cells were seeded on the surface of MatTek glass bottom microwell dishes using 1 mL media. After 1 day, the cells were washed twice with warm DMEM media, incubated at 37 °C with 2 µM peptide 9k for 90 min and fixed. Images were taken using a Leica TCS SP8 confocal fluorescence microscope.
5
Vinculin and Actin Staining on PDMS
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NG108-15 cells were grown on PDMS (covered with or without Au NP film), washed in DPBS, fixed with 4% paraformaldehyde for 15 min, treated with 0.25% Triton X-100 for 10 min, and rinsed with DPBS. The cells were incubated with 1.2% bovine serum albumin for 2 h at 37 °C. To stain for vinculin, the cells were incubated with 2 μg/mL anti-vinculin antibody in DPBS with 1% bovine serum albumin for 3 h at room temperature. The samples were then washed three times with ice-cold DPBS, and a goat anti-rabbit IgG (H + L) secondary antibody (2 μg/mL) was added and incubated for 30 min at room temperature. The samples were then washed with ice-cold DPBS. To stain for actin, ActinRed™ 555 ReadyProbes® Reagent (diluted 10-fold in DPBS) was added. After incubation for 30 min at room temperature, the samples were washed again with ice-cold DPBS, stained with DAPI (1/100 in DPBS), and thoroughly washed. Confocal imaging was performed with a Leica TCS SP8 confocal fluorescence microscope. DAPI, green fluorescent protein, and red fluorescent protein filters were used to detect the cell nuclei, vinculin, and actin, respectively.
6
Immunofluorescence Staining of Cells
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Vero and HEK-293T Cell monolayers were fixed using 4% paraformaldehyde (AAPR12-500, Python bio) for 15 min at room temperature. Then, cells were permeabilized with 0.1% Triton-X 100 (AAPR96-C100, Python bio) for 15 min at room temperature. Next, we block it with 5% skimmed milk powder at 4 °C overnight. After that, cells were incubated with primary antibodies for 1 h at room temperature, then the fluorochrome-conjugated secondary antibodies for 1 h in the dark. Last, cells were incubated with DAPI (P36941, Invitrogen) for nuclear staining. The fluorescence signals were detected with a TCS SP8 confocal fluorescence microscope (Leica).
7
Laser-treated NG108-15 Cell Viability
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NG108-15 cells subjected to laser treatment were stained with the LIVE/DEAD Cell Imaging Kit (diluted 1/2 in DPBS) for 20 min at room temperature. After staining, the cells were washed three times with ice-cold DPBS. Confocal images were taken with a Leica TCS SP8 confocal fluorescence microscope, with excitation at 488 and 552 nm.
8
Confocal Microscopy of P. salmonis
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The acquisition settings were the same in all treatments for the three independent plates. Images were taken using a TCS SP8 confocal fluorescence microscope (Leica, Wetzlar, Germany) with numerical aperture 1.4 × 100 oil objective, ×2 digital zoom (NA = 1.4; HC PL APO CS2 100×) and a z-step of 1 μm optical sections (velocity scan 600 Hz; resolution 1024 × 1024 pixels, equivalent to 58.13 μm × 58.13 μm). The following three laser wavelengths were used for DAPI (Ex 405 nm and Em 410–483 nm), FITC (Ex 488 nm and Em 502–621 nm), and Alexa Fluor® 647 (Ex 638 nm and Em 650–776 nm), and signals were detected with an ultrahigh dynamic photomultiplier (PMT) spectral detector. Maximum intensity projections of confocal z-stack images of P. salmonis in whole nuclei (containing 17–22 stacks with resolution equivalent to 4.81–6.21 μm) were analyzed. All pictures and three-dimensional (3D) reconstructions of myotubes were performed using Leica Application Suite X v.3.5.5 software (Leica).
9
Nanomaterial-Mediated Radiosensitization of 4T1 Cells
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4T1 cells were seeded in glass-bottomed Petri dishes at an initial density of 2 × 104 cells/dish overnight and incubated with nanomaterials Au4-IO NP/Au4-IO NP-cRGD of 2 μM for 6 h followed by irradiation at 0 and 4 Gy X-ray. After 24 h, the cells were stained with the CellEvent™ Caspase-3/7 Green Reagent. Confocal images were taken with a Leica TCS SP8 confocal fluorescence microscope (excitation: 488 nm).
10
Immunofluorescence Assay for γ-H2AX
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4T1 cells were cultured on glass-bottomed petri dishes for incubation of materials and X-ray treatment. 24 h later, the cells were fixed with 4% paraformaldehyde and permeated with 0.25% Triton X-100. Then, the cells were blocked with 1% bovine serum albumin (BSA) for 1 h and incubated with γ-H2AX antibody (MA5-33062, Invitrogen) overnight at 4 °C. Ultimately, the secondary antibody Alexa Fluor™ 647 tag goat anti-rabbit IgG (H + L) (A21245, Invitrogen) was added for 1 ~ 2 h, and DAPI was used to stain cell nuclei. The cells were imaged using Leica TCS SP8 confocal fluorescence microscope at excitation wavelengths of 405 and 647 nm.
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