The cells were cultured on coverslips, which were kept in a 35-mm Petri dish for 16–20 h before treatment. After treatment with or without FND (50 μg/ml for 24 h), the cells were washed with isotonic PBS (pH 7.4), and then were fixed with 4% paraformaldehyde solution in PBS for 1 h at 37°C. The coverslips were washed three times with PBS, and non-specific binding sites were blocked in PBS containing 10% FBS, 0.3% Triton X-100 for 1 h. The cells were incubated with mouse anti-SSEA-1 (1:100) and rabbit anti-β-III-tubulin antibodies (1:100) in PBS containing 10% FBS for overnight at 4°C. Thereafter, the cells were washed three times with 0.3% Triton X-100 in PBS. Subsequently, the cells were incubated with goat anti-mouse Cy3 (1:100) and anti-rabbit Hylite 488 (1:100) in PBS containing 10% FBS for 1 h at 37°C in dark. The nuclei or chromosomes were stained with Hoechst 33258. After staining, the samples were examined under a Multiphoton Confocal Microscope System (TCS-SP5-X AOBS, Leica, Germany).
Multiphoton confocal microscope system tcs sp5 x aobs
Manufactured by Leica
Sourced in Germany
The Multiphoton Confocal Microscope System (TCS-SP5-X AOBS) is a high-performance microscope designed for advanced imaging applications. It features a combination of confocal and multiphoton technologies, allowing for the capture of detailed, high-resolution images of biological samples. The system is equipped with various laser sources and a precisely controlled scanning mechanism to enable advanced imaging techniques.
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2 protocols using multiphoton confocal microscope system tcs sp5 x aobs
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Immunofluorescence Staining of Stem Cells
2
Immunofluorescence Analysis of Phosphorylated Histone H3
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The cells were cultured on coverslips, which were kept in 35-mm Petri dish at a density of 5 × 105 per well for 16–20 h. After treatment with or without ND-conjugates, the cells were washed with PBS. The cells were fixed with 4% paraformaldehyde solution overnight at 4 °C. Then the cells were washed three times with PBS and non-specific binding sites were blocked in PBS containing 10% FBS and 0.3% Triton X-100 for 1 h at 37 °C. Thereafter, the cells were separately incubated with rabbit anti- p-H3S10 (1:100) antibody in PBS containing 10% FBS overnight at 4 °C, and washed three times with 0.3% Triton X-100 in PBS. Then the cells were incubated with anti-rabbit IgG-Hylite 488 (1:100) in PBS containing 10% FBS for 1 h at 37 °C, and washed three times with 0.3% Triton X-100 in PBS. The β-tubulin and nuclei were stained with the Cy3-labeled anti-β-tubulin (1:100) and Hoechst 33258 (Sigma Chemical Co., St. Louis, Mo), respectively. The samples were examined under a Multiphoton Confocal Microscope System (TCS-SP5-X AOBS, Leica, Germany).
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