The intra-oocyte GSH contents of IVM oocytes were measured by applying previously described methods [17 (link)23 (link)]. Briefly, CellTracker Blue 4-chloromethyl-6.8-difluoro-7-hydroxycoumarin (Invitrogen) was used to detect intra-oocyte GSH based on blue fluorescence. From each treated group, denuded oocytes having visually extruded first polar body were incubated in the dark for 30 min in TLH-PVA supplemented with 10 μM CellTracker, followed by incubation for an additional 30 min in IVC medium. The oocytes were subsequently washed with Dulbecco’s phosphate-buffered saline (Invitrogen) containing 0.1% (w/v) PVA, and placed in 2-µL micro-droplets. Fluorescence was observed under an epifluorescence microscope (TE300; Nikon) with ultraviolet ray filters at 370 nm. The fluorescence intensity of each oocyte was analyzed using the ImageJ software (version 1.49q; National Institutes of Health), and normalized to that of oocytes matured in standard M199.
Te300 epifluorescence microscope
Manufactured by Nikon
Sourced in Japan
The TE300 is an epifluorescence microscope manufactured by Nikon. It is designed to facilitate the observation and analysis of fluorescently labeled specimens. The microscope features a high-performance optical system and a range of illumination options to support various fluorescence imaging techniques.
Automatically generated - may contain errors
Lab products found in correlation
Celltracker blue 4 chloromethyl 6.8 difluoro 7 hydroxycoumarin, by Thermo Fisher Scientific (1 mentions)
Dulbecco s phosphate buffered saline, by Thermo Fisher Scientific (1 mentions)
Digital sight ds qi1mc camera, by Nikon (1 mentions)
Csu 10 spinning disk confocal unit, by Hamamatsu Photonics (1 mentions)
5 protocols using te300 epifluorescence microscope
1
Measuring Intracellular Glutathione in IVM Oocytes
2
Melatonin's Effect on Embryo ROS
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A total of 306 embryos were used to explore the effect of melatonin on intracellular ROS. Embryos were cultured in M16 medium supplemented with different concentrations of melatonin (0–10−3 M). After 24 h, the embryos at the 2–4 cell stage were collected to detect their intracellular ROS levels. Briefly, 2′,7′-dichlorohydrofluorescein diacetate (DCHFDA) (Beyotime Institute of Biotechnology, Jiangsu, China) was used as a green fluorescence indicator to detect intracellular ROS. A total of 25–30 embryos from each treatment group were incubated in DPBS-0.1% PVA containing 10 μM DCHFDA in the dark environment for 30 min, and the embryos were then washed three times with DPBS-0.1% PVA. The green fluorescence was measured using an epifluorescence microscope (TE300, Nikon, Tokyo, Japan) with UV filters (460 nm for ROS). The fluorescent images were saved as graphic files in TIF format. The fluorescence intensities of the embryos were analyzed using the ImageJ software (Version 1.40, National Institutes of Health, Bethesda, MD, USA) and normalized to that of the control embryos.
3
Fluorescent Microscopy Imaging of Stained Cells
4
Live Cell Microscopy Protocol
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Live cell microscopy was performed on a Carl Zeiss 200 m inverted microscope equipped with a Yokogawa CSU-10 spinning disk confocal unit, 100 × objective, 488 and 561 nm excitation lasers, and a Hamamatsu C9100-13 EMCCD detector. Any image panel featuring DAPI staining used fixed cells and was collected on a Nikon TE300 epifluorescence microscope equipped with a Nikon digital sight DS-Qi1MC camera using a 100 × objective and illuminated by LED. DAPI stained cells were prepared by inoculating 1 mL of YPD medium with cells from a yeast colony and incubating overnight. Cells were then sedimented by centrifugation at 3,000 g and resuspended in 100 μL of phosphate-buffered saline containing 4% formaldehyde. Fixation proceeded at room temperature for 15 minutes, after which cells were sedimented, washed with 1 mL tris-buffered saline (TBS), and again resuspended in 100 μL of a TBS solution containing 1% Triton X-100 and 200 ng/mL DAPI and incubated at room temperature for 30 minutes. Cells were again washed in 1 mL TBS before spotting on glass slides and imaging.
5
Evaluating Blastocyst Development in IVF/SCNT
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Day 0 was regarded as the day on which IVF or SCNT was initiated. On day 2, the CL rates of embryos were evaluated to assess their developmental competence, and the embryos were transferred to fresh PZM3 droplets. The normally cleaved embryos were classified into three groups, 2–3, 4–6, and 6–8 cells, while one-cell and fragmented embryos were excluded. On day 4, 10% FBS was added to the PZM3 droplets containing embryos. Finally, on day 7, the BLs formed were categorized into three groups as follows: early, expanded, and hatched BLs. All BLs from each group were collected, and the ZP was removed (except for hatched BLs) with 0.5% protease. Thereafter, the zona-free BLs were fixed with 3.7% paraformaldehyde in DPBS containing 0.1% PVA for 5 min and washed with DPBS containing 0.1% PVA. The fixed BLs were stained with 10 μg/ml Hoechst 33342 for 5 min, then mounted on glass slides in 100% glycerol droplets, and gently covered with coverslips. The slides were observed under a TE300 epifluorescence microscope (Nikon) with an ultraviolet filter (370 nm).
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