EdU incorporation was used to assess the percentage of cells in S-phase of the cell cycle. Briefly, live zebrafish larvae (7 dpf) were incubated at 28°C in 2 mM EdU (Invitrogen, #C10340) in E3 medium for 2 hr followed by a further incubation in fresh E3 medium for 1 hr. Larvae were euthanised using benzocaine (1000 mg/L; Sigma, #PHR1158) prior to removal of the liver by micro-dissection. EdU labelling was carried out using the Click-iT Edu Alexa Fluor 647 (AF647) imaging kit (Invitrogen, #C10340), according to the manufacturer’s instructions. The livers were co-stained with Hoechst 33342 (Thermo Fisher, #62249). Image acquisition was performed using an Olympus FVMPE-RS multiphoton microscope with excitation wavelengths of 950 nm for Hoechst 33342 dye and 1160 nm for AF647 (Thermo Fisher, #A21235). The number of Hoechst 33342- and EdU-positive cells was quantified using Arivis Vision4D software.
Fvmpe rs multiphoton microscope
Manufactured by Thermo Fisher Scientific
The FVMPE-RS multiphoton microscope is a high-performance imaging system designed for advanced cellular and tissue analysis. It utilizes multiphoton excitation technology to enable deep tissue imaging with reduced phototoxicity and improved signal-to-noise ratio. The core function of the FVMPE-RS is to provide researchers with a powerful tool for visualizing and analyzing complex biological samples in a non-invasive manner.
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2 protocols using fvmpe rs multiphoton microscope
1
Quantifying Cellular Proliferation in Zebrafish Livers
2
Measuring Cell Cycle Dynamics in Zebrafish Liver
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EdU incorporation was used to assess the percentage of cells in S phase of the cell cycle. Briefly, live zebrafish larvae (7 dpf) were incubated at 28 o C in 2 mM EdU (Invitrogen, #C10340) in E3 medium for 2 h followed by a further incubation in fresh E3 medium for 1 h. Larvae were euthanised using benzocaine (1000 mg/L; Sigma, #PHR1158) prior to removal of the liver by micro-dissection. EdU labelling was carried out using the Click-iT Edu Alexa Fluor 647 (AF647) imaging kit (Invitrogen, #C10340), according to the manufacturer's instructions. The livers were co-stained with Hoechst 33342 (Thermofisher, #62249). Image acquisition was performed using an Olympus FVMPE-RS multiphoton microscope with excitation wavelengths of 950 nm for Hoechst 33342 dye and 1160 nm for AF647 (Thermofisher, #A21235). The number of Hoechst 33342 and EdU positive cells was quantified using Arivis Vision4D software.
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