HLE, Bel 7402, and PLC/PRF/5 cells were stained as described previously [19 (link)]. To observe the migration of caspase-3 molecules, cells were treated with Vinco (10 µg/mL and 80 µg/mL); to evaluate the expression and location of Src, Oct4, and phosphate and tension homology deleted on chromosome 10 (PTEN); cells were treated with Vinco (80 µg/mL). Laser confocal microscopy was performed to observe the expression and location of the target proteins. Briefly, cells were fixed in 4% paraformaldehyde and incubated with mouse anti-human caspase-3, anti-Src antibodies, rabbit anti-human Oct4, anti-PTEN antibodies (Abcam Trading (Shanghai) Company, Ltd., Shanghai, China) for 24 h. Alex488-, Alex647-, or fluorescein isothiocyanate (FITC)-conjugated secondary anti-mouse immunoglobulin G (IgG) was added, and the cells were incubated for 2 h. Afterwards, 100 µL of DAPI (1 µg/mL) was added for 30 min. The cells were visualized with a Leica TCS-NT SP2 laser confocal microscope (Leica Camera, Wetzlar, Germany).
Tcs nt sp2 laser confocal microscope
Manufactured by Leica
Sourced in Germany
The TCS-NT SP2 is a laser confocal microscope manufactured by Leica. It is designed to capture high-resolution, three-dimensional images of biological samples. The microscope utilizes a laser light source and a confocal scanning system to produce optical sections, allowing for detailed analysis of complex structures within the sample.
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5 protocols using tcs nt sp2 laser confocal microscope
1
Visualizing Caspase-3 and Src Localization
2
Visualizing Caspase-3 and Signaling Proteins
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HLE, Bel 7402 and PLC/PRF/5 cells were stained as described previously [Li et al., 2013] . To observe the migration of caspase-3 molecules, cells were treated with Vinco(10 µg/ml and 80 µg/ml); to evaluate the expression and location of Src, Oct4 and PTEN, cells were treated with Vinco(80 µg/ml). Laser confocal microscopy was performed to observe the expression and location of the target proteins. Brie y, cells were xed in 4% paraformaldehyde and incubated with mouse anti-human caspase-3, anti-Src antibodies, rabbit anti-human Oct4, anti-PTEN antibodies(Abcam Trading (Shanghai) Company, Ltd., Shanghai, China) for 24 h. Alex 488-, Alex 647-, or uorescein isothiocyanate (FITC)-conjugated secondary antimouse immunoglobulin G (IgG) was added, and the cells were incubated for 2 h. Afterwards, 100 µl of DAPI (1 µg/ml) was added for 30 min. The cells were visualized with a Leica TCS-NT SP2 laser confocal microscope(Leica Camera, Wetzlar, Germany).
3
Caspase-3 Activation in Liver Cancer Cells
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Bel 7402 cells and HLE cells were stained as described previously [21 (link)]. Briefly, cells were fixed in 4% paraformaldehyde and incubated with mouse anti-human caspase-3 antibody for 12 h. Fluorescein isothiocyanate (FITC)-conjugated secondary anti-mouse immunoglobulin G was added and incubated for 2 h, followed by the addition of 100 μL DAPI (1 μg/mL) for 30 min. Cells were visualized with a Leica TCS-NT SP2 laser confocal microscope (Leica Camera, Wetzlar, Germany).
4
FRET Assay for AFP-PTEN Interaction
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Fluorescence resonance energy transfer was employed to investigate the interaction between AFP and PTEN in L-02, L-02-X, CHL, CHL-X, PLC/PRF/5, and HLE cell lines as previously described [36 (link)]. Briefly, cells were fixed in paraformaldehyde (4%), and rabbit anti-PTEN antibody and mouse anti-human AFP antibody (Santa Cruz Biotechnology) were added and incubated for 12 hours. Secondary goat anti-mouse or anti-rabbit immunoglobulin G conjugated with fluorescence isothiocyanate (FITC) or tetramethylrhodamine isothiocyanate (Zhongshan Biol Tech Co., Beijing) was applied for 2 hours, followed by the addition of 100 μL of 4′,6-diamidino-2-phenylindole (DAPI) (1 μg/mL). Cells were visualized with the Leica TCS-NT SP2 laser confocal microscope and associated software (Leica Camera) and analyzed by fluorescence resonance energy transfer.
5
p-mTOR Immunofluorescence Staining Protocol
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The staining procedure for confocal microscopy has been previously described [35 (link)]. Briefly, cells were fixed in 4% paraformaldehyde and incubated with rabbit anti-human p-mTOR (Ser2448) antibody for 12 hours. FITC-conjugated secondary anti-rabbit immunoglobulin G was added and incubated for 2 hours, followed by the addition of 100 μL DAPI (1 μg/mL) and 30 minutes of incubation. Cells were visualized with the Leica TCS-NT SP2 laser confocal microscope (Leica Camera).
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