S100tv inverted microscope

Mito-GFP mRNA was transcribed using the T3 mMessage mMachine Kit (Ambion) according to the manufacturer’s protocol and resuspended in RNase-free water. mRNA (1.2 mg/ml) was delivered using a microinjection apparatus consisting of Narishige micromanipulators (Narishige, Japan) mounted on a Zeiss S100TV inverted microscope. A controlled delivery of ∼5% oocyte volume was delivered to GV stage-arrested oocytes using a picopump. After injection, oocytes were washed out of 3-isobutyl-1-methylxanthine (IBMX) and transferred in M16 medium. To label the endoplasmic reticulum, 1,1′-Dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI, Sigma-Aldrich) was microinjected as a saturated solution in soybean oil (Sigma-Aldrich) 30 min prior to imaging. DiI was excited using the 552 nm laser, and fluorescence collected using a 560–630 nm band pass filter. Mito-GFP was excited using the 488 nm laser and fluorescence collected using a 495–540 nm band pass filter.

Cyclin A2–GFP mRNA (a gift from J. Pines, The Institute of Cancer Research, London, England, UK) was transcribed using the T7 mMessage mMachine kit (Ambion) according to the manufacturer’s protocol and resuspended in RNase-free water. mRNA (1.2 mg/ml) was delivered using a microinjection apparatus consisting of Narishige micromanipulators mounted on a Zeiss S100TV inverted microscope. A controlled delivery of ∼5% oocyte volume was delivered to GV stage–arrested oocytes using a picopump. After injection, oocytes were washed out of IBMX and transferred in M16 medium. For Western blot, the injected GV oocytes were arrested for 2 h (imitating the time from GV to GVBD) in 200 µM IBMX for the expression of mRNA before collection.