Nikon Diaphot ECLIPSE TE 300 (Nikon UK Ltd) was used to perform microinjections of oocytes within 30 min. A volume of 20 μM Sept 9 siRNAs or control siRNA from JTSBIO Co., Ltd was used for microinjection into the cytoplasm of oocytes to deplete Septin 9 and as a control. A volume of 20 μM Cdc20 siRNA from JTSBIO Co., Ltd was microinjected into the cytoplasm of oocytes to deplete CDC20. The same amount of control siRNA was injected as a control, as described in a previous report.40 After injection, the GV oocytes were arrested at the GV stage in M2 medium with 200 μM IBMX for 24 h to allow depletion of Septin 9 or CDC20. Next, we fully washed the oocytes and transferred them into IBMX‐free medium. Next, to examine the expression level of Septin 9‐mCherry, Myc‐CDC20 or CCNB1‐GFP dynamics and trace the temporal and spatial extrusion of the first polar body (PBE), 20 ng/μl Sept 9 mRNA or Cdc20 mRNA, CCNB1 mRNA (40 ng/μl); MAP4‐GFP mRNA (200 ng/μl) and H2B‐mcherry mRNA (50 ng/μl) was injected into the GV oocytes. Each oocyte was microinjected with approximately 10 pl of Sept 9 siRNA, Cdc20 siRNA, Sept 9 mRNA, Cdc20 mRNA or control siRNA or MAP4‐GFP mRNA or H2B‐mcherry mRNA and control mRNA. Each experiment was performed three times separately and no less than 150 oocytes were used in each group.
Diaphot eclipse te 300
Manufactured by Nikon
Sourced in United Kingdom
The Nikon Diaphot ECLIPSE TE 300 is an inverted microscope designed for a variety of laboratory applications. It features a sturdy, stable frame and is equipped with high-quality optical components for clear, detailed observations.
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6 protocols using diaphot eclipse te 300
1
Septin 9 and CDC20 Depletion in Oocytes
2
Microinjection of dsRNA into Oocytes
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Microinjection of JMY dsRNA into the cytoplasm of oocytes was performed as described previously [18] (link) with the Femtojet constant flow system (Eppendorf AG, Hamburg, Germany) and a Nikon Diaphot ECLIPSE TE300 inverted microscope (Nikon UK Ltd., Kingston upon Thames, Surrey, UK) equipped with a Narishige MM0-202N hydraulic three-dimensional micromanipulator (Narishige Inc., Sea Cliff, NY, USA). Each oocyte was injected with approximately 10 pL (1 µg/uL) of JMY dsRNA, and oocytes were cultured under paraffin oil at 38.5°C. The developmental stage of oocytes was determined by staining with 1 µg/mL of 4′-6-diamidino-2-phenylindole (DAPI) for 10 min. The control oocytes were microinjected with 5–10 picoliter of green fluorescent protein dsRNA. All microinjection experiments were performed at least five independent times, and approximately 100 oocytes were injected in each group.
3
siRNA Microinjection in Oocytes and Embryos
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SiRNAs were prepared as described above and then microinjected into the cytoplasm of MII oocyte, zygote, or single blastomere of 2-cell embryos as previously described[22] (link), and according to our previous work[23] (link),[24] (link). The microinjections were repeated at least three times, and at least 100 oocytes or embryos were used. Nikon Diaphot Eclipse TE 300 inverting microscope (Nikon, Yuko, Japan), equipped with Narishige MM0-202N hydraulic three-dimensional micromanipulators (Narishige Inc., Tokyo, Japan), was used in these experiments. Approximately 10 pL diluted siRNA was injected into one oocyte or embryo in all experiments. After microinjection, MII oocytes or embryos were washed thoroughly and cultured in CZB medium under mineral oil at 37°C in a 5% CO2 atmosphere, and observed at specific stages of development.
4
Mitochondria Microinjection in Oocytes
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Mitochondria were prepared by differential centrifugation. The ADSCs were prepared with HTF at 107 cells per ml. Then the cells were treated 4-6 times with a cell homogenizer on ice. The homogenate was centrifuged for 20 min at 2000r/m, the supernatant was collected and centrifuged for 20 min at 10000r/m again. The sediments were resuspended very slowly with 1μL HTF. That is, the mitochondria extracted from 107 ADSCs were in 1uL HTF solution. All the processes were carried out at 4°C, and the extracts were maintained at 4°C for microinjection. For the GV oocytes, 7-8pL mitochondrial in HTF were micro-injected into each oocyte using a Nikon Diaphot ECLIPSE TE 300 (Nikon UK Ltd., UK) inverted microscope equipped with Narishige MM0202N hydraulic three-dimensional micromanipulator (Narishige Inc., USA) and microinjection was completed within 30 minutes. For MII oocytes, 10pL mitochondria were microinjected into each oocyte combing ICSI (for details, please see “ICSI and embryo transfer” below).
5
Microinjection and Live Imaging of GV Oocytes
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Microinjection of Rac3 SiRNAs (Gene Pharma, Shanghai, China) or Alexa 488-phalloidin (Invitrogen, Carlsbad, CA, USA) into GV oocytes was performed using a Nikon Diaphot ECLIPSE TE 300 (Nikon UK Ltd., Kingston upon Thames, Surrey, UK) inverted microscope equipped with Narishige MM0202N hydraulic three-dimensional micromanipulators (Narishige Inc., Sea Cliff, NY, USA) and completed within 30 minutes.
Rac3 SiRNAs were microinjected into GV oocyte to knockdown Rac3. The subsequent sequences of Rac3 siRNAs were used at 20 μM each, Rac3 siRNA-1:5’- GGAAGACUACGAUAGGCUUTT-3’ ;
Rac3 siRNA-2:5’- GCAAGAAGUGCACUGUAUUTT-3’ ;
Rac3 siRNA-3:5’—GCCUUCCCAGGAGAAUAUATT-3’ .
The same amount of negative control siRNAs were injected as control. Microinjected oocytes were cultured in M16 medium with 100μM IBMhX for 24 h. Then the oocytes were cultured in M16 medium.
For live oocyte imaging, after 1–2 hours of culture, the microinjected oocytes cultured in M16 medium with 10ng/mL Hoechst 33342 were used for live oocyte imaging on a Perkin Elmer precisely Ultra VIEW VOX confocal Imaging System (PerkinElmer, Waltham, MA, USA).
Rac3 SiRNAs were microinjected into GV oocyte to knockdown Rac3. The subsequent sequences of Rac3 siRNAs were used at 20 μM each, Rac3 siRNA-1:
Rac3 siRNA-2:
Rac3 siRNA-3:
The same amount of negative control siRNAs were injected as control. Microinjected oocytes were cultured in M16 medium with 100μM IBMhX for 24 h. Then the oocytes were cultured in M16 medium.
For live oocyte imaging, after 1–2 hours of culture, the microinjected oocytes cultured in M16 medium with 10ng/mL Hoechst 33342 were used for live oocyte imaging on a Perkin Elmer precisely Ultra VIEW VOX confocal Imaging System (PerkinElmer, Waltham, MA, USA).
6
Kif2a Knockdown in Oocyte Maturation
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Microinjections were performed using a Narishige microinjector under a Nikon Diaphot ECLIPSE TE300 (Nikon UK Ltd.) and completed within 30 min. Small interfering RNAs (siRNAs) of Kif2a (Gene Pharma) were microinjected into the cytoplasm to knockdown Kif2a. The subsequent sequences of Kif2a siRNAs were used at 20 μM each, Kif2a siRNA-1: 5′-GGCCACUAGUGGAAACAAUTT-3′; Kif2a siRNA–2: 5′-GGAUGUUGAUGCUACAAAU TT-3′; Kif2a siRNA–3: 5′-GAAAACGACCACUCAAUAATT-3′. The same amount of negative control siRNAs was also injected as control. Then, oocytes were arrested at the GV stage in M2 medium supplemented with 100 μM IBMX for 24 h to knockdown Kif2a. Then the oocytes were thoroughly washed and transferred into IBMX-free M2 medium for further culture.
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