Immunolabeling and Live-cell Imaging of Oocytes

Oocytes or oocytes were fixed in a solution of 4% paraformaldehyde and 2% triton X-100 in PBS for 30 min at room temperature. Blocking was performed in PBS with 10% goat or donkey serum, 10% BSA and 2% Tween for 60 min at room temperature. Antibodies used for immuno-labelling: pPlk1 (1 : 200, Santa Cruz Biotechnology), N-Wasp (1 : 100, Cell Signaling Technology), TACC3 (AF5720-SP; R&D System), rat anti-tyrosinated α-tubulin (1 : 1000, Serotec, YL1/2) and FITC-conjugated mouse anti-α-tubulin (1 : 200, Alexa Fluor 488, Invitrogen) and detected with secondary antibody of Goat anti-Rabbit IgG antibody (1 : 1000, Alexa Fluor 555, Invitrogen). Phalloidin Alexa Fluor 555 (1 : 20, Cell Signaling Technology) was used where appropriate to stain actin. DNA was labelled using 10 min incubation in Hoechst 33 342 (10 µg ml⁻¹, Sigma-Aldrich). Serial Z sections of fixed oocytes/oocytes were acquired at room temperature in a glass-bottomed dish using laser-scanning confocal microscope imaging system (SP8, Leica). For live-cell imaging, the microinjected oocytes were incubated in M2 media at 37°C under mineral oil and imaged with Leica SP8 confocal microscope. In some live imaging experiments, chromosomes were labelled with 1 µM Sir-DNA (Spirochrome)