Cells seeded onto pNFSs were fixed with 3.7% PFA for 15 min, washed three times with PBS, and permeabilized with 0.1% (v/v) Triton X-100. After 15 min, the pNFSs were washed three times with PBS and incubated in a blocking solution (PBS/3% BSA) for 1 h at room temperature. The pNFSs were then incubated overnight at 4 °C with an appropriate primary antibody. Thereafter, the pNFSs were washed three times with PBS and incubated with Alexa Fluor 594-conjugated secondary antibodies for 1 h. The pNFSs were washed twice with PBS and cell nuclei were counterstained with DAPI (Invitrogen, Carlsbad, CA, USA) for 2 min. The pNFSs were washed three times with PBS, mounted onto slides using mounting reagent (Invitrogen), and analyzed using a C1-Plus laser-scanning TE2000E confocal microscope (Nikon, Tokyo, Japan).
C1 plus laser scanning te2000e confocal microscope
Manufactured by Nikon
Sourced in Japan
The C1-Plus laser-scanning TE2000E confocal microscope is a high-performance imaging system designed for advanced microscopy applications. It features a TE2000E inverted microscope platform and integrates a confocal scanning unit, multiple laser sources, and a high-sensitivity detector system. The core function of this microscope is to provide high-resolution, optically sectioned imaging of samples through the use of a focused laser beam and a pinhole aperture to reject out-of-focus light.
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7 protocols using c1 plus laser scanning te2000e confocal microscope
1
Immunofluorescence Analysis of pNFSs
2
Multi-layer pNFS Cell Culture Protocol
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Cells were seeded onto pNFS and incubated in a humidified 5% CO2/95% air incubator at 37 °C. After 3 days, pNFSs were stacked in four layers and incubated for 3 days. Twenty-four hours prior to separation, 10 μM BrdU was added to the multi-layered pNFS. Upon separation, each layer was washed three times with PBS and fixed with 3.7% PFA for 15 min. The cells were then washed three times with PBS, permeabilized with 0.1% (v/v) Triton X-100, and treated with 2 N HCl for 10 min. After washing three times with PBS, cells were incubated with a primary antibody against BrdU, followed by incubation with secondary antibody and counterstaining with DAPI. Images were obtained using a C1-Plus laser-scanning TE2000E confocal microscope (Nikon, Tokyo, Japan).
3
Fluorescent Staining of Cells on pNFS
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Cells were seeded onto pNFS and incubated in a humidified 5% CO2/95% air incubator at 37 °C. After incubating for 3 days, cells were fixed with 3.7% PFA for 15 min. Thereafter, cells were washed three times with PBS, permeabilized with 0.1% (v/v) Triton X-100, washed three times with PBS, and incubated in blocking solution (3% bovine serum albumin (BSA)) for 1 h at room temperature. The pNFSs were then incubated overnight at 4 °C with Alexa Fluor 594 phalloidin. Thereafter, the pNFSs were washed twice with PBS, and cell nuclei were stained with DAPI (Invitrogen, Carlsbad, CA, USA) for 2 min. The pNFSs were washed three times with PBS, mounted onto slides using mounting reagent (Invitrogen, Carlsbad, CA, USA), and analyzed using a C1-Plus laser-scanning TE2000E confocal microscope (Nikon, Tokyo, Japan).
4
Cell Viability in Layered pNFS
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Cells were seeded onto pNFSs and incubated in a humidified 5% CO2/95% air incubator at 37 °C. After 3 days, pNFSs were stacked in four layers and incubated for 3 days. During the latter 3-day incubation period, designated samples of 4-layer pNFSs were separated into individual layers on each day, and cells in each layer were analyzed for viability using a LIVE/DEAD Fixable Green Dead Cell Stain Kit (Thermo Fisher Scientific, Waltham, MA, USA). The fluorescence of dead (green) cells was analyzed using a C1-Plus laser-scanning TE2000E confocal microscope (Nikon, Tokyo, Japan).
5
Hypoxia Analysis in 3D Cell Culture
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Cells were seeded onto pNFS and incubated in a humidified 5% CO2/95% air incubator at 37 °C. After culturing cells on individual pNFSs for 3 days, pNFSs were stacked into four layers and incubated for an additional 3 days. Six hours prior to separation, pimonidazole (50 μM) was added. Upon separation, individual layers were washed three times with PBS, fixed with 3.7% PFA for 15 min, then washed again and permeabilized with 0.1% (v/v) Triton X-100. After washing three times with PBS, cells were incubated with primary antibody against pimonidazole followed by incubation with secondary antibody and counterstaining with DAPI. Images were obtained using a C1-Plus laser-scanning TE2000E confocal microscope (Nikon, Tokyo, Japan).
6
In Situ Apoptosis Detection in OGD-Exposed Cells
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Cells grown on 8-well chamber slides were exposed to OGD. After fixing with 4% (v/v) paraformaldehyde for 15 min, cells were washed with PBS containing 1% (w/v) bovine serum albumin, permeabilized with 0.1% (v/v) Triton-X100, washed with PBS, and incubated for 1 h at 37°C in the dark with an apoptosis detection solution (Apoptosis Detection System kit; Roche Molecular Biochemicals, Mannheim, Germany). In situ-labeled nuclei were observed and photographed using a Nikon C1-Plus laser-scanning TE2000-E confocal microscope.
7
Apoptosis Induction and Quantification
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Cells were seeded onto pNFS and incubated in a humidified 5% CO2/95% air incubator at 37 °C. After 3 days, pNFSs were stacked in four layers and incubated for 1 day, after which cells were treated with 3 μM DOX or exposed to ionizing radiation (4 Gy). After 1 day, cells were fixed with 3.7% PFA for 15 min, washed with PBS containing 3% (w/v) BSA, and permeabilized with 0.1% (v/v) Triton X-100. After washing with PBS, cells were incubated for 1 h at 37 °C in the dark with an apoptosis-detection solution (Apoptosis Detection System Kit; Roche Molecular Biochemicals, Mannheim, Germany). In situ-labeled nuclei were observed and imaged using a C1-Plus laser-scanning TE2000E confocal microscope (Nikon, Tokyo, Japan).
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