Confocal microscopy

For single staining of Kif4a, ac-tubulin, α-tubulin, Crest staining, oocytes were fixed in 4% paraformaldehyde (in PBS) at room temperature for 30min and then permeabilized with 0.5% Triton X-100 in PBS for 20min. To reduce non-specific IgG binding, oocytes were blocked in blocking buffer (1% BSA-supplemented PBS) at room temperature for 1 h. For Kif4a or ac-tubulin staining, oocytes were incubated with a rabbit polyclonal anti-Kif4a antibody (1:50) or a mouse monoclonal anti- ac-tubulin antibody (1:100) at 4 °C overnight or at room temperature for 4 h. For Crest staining, oocytes were incubated with a Human anti-centromere CREST (1:200) at 4 ? for 48 h. After 3 washes (2 min each) with wash buffer (0.1% Tween 20 and 0.01% Triton X-100 in PBS), oocytes were labeled with an appropriate secondary antibody coupled to FITC-conjugated and TRITC-conjugated goat anti-rabbit IgG (1:100; for Kif4a staining), TRITC-conjugated goat-anti-mouse IgG (1:100; for ac-tubulin staining) or TRITC-donkey-anti-Human IgG at room temperature for 1 h. For α-tubulin-FITC staining, oocytes were incubated with an anti-α-tubulin-FITC antibody (1:100) at room temperature for 4 h. After 3 washes in wash buffer, oocytes were co-stained with DAPI to examine chromosomes. After staining, samples were mounted on glass slides and observed with a confocal laser-scanning microscope (Zeiss LSM 700 META, Germany). Furthermore, ImageJ software (National Institutes of Health, Bethesda, MD, USA) was used to quantify the intensity of fluorescence.