Anti bubr1

Unless otherwise specified, all chemicals and reagents were purchased from Sigma-Aldrich (United States). Antibodies used in this study include: rabbit polyclonal anti-EML1 (1:100, Proteintech, #12765-AP), rabbit polyclonal anti-EML4 (1:500, Cell Signaling Technology, #2428), rabbit polyclonal anti-Gamma Tubulin (1:200, Proteintech, #15176-AP), rabbit polyclonal anti-Pericentrin (1:200, Abcam, # ab4448), rabbit polyclonal anti-Phospho-cdc2 (Tyr15) (1:500, CST, #9111); rabbit monoclonal anti-BubR1(1:250, Abcam, #ab3305), rabbit polyclonal anti-tRFP/mKate (1:500, evrogen, #AB234), mouse monoclonal anti-Cyclin B1 (1:100, abcam, #ab72), mouse monoclonal anti-Cdk1/Cdk2 (1:100, Santa Cruz, #sc-53219), mouse monoclonal anti-NUDC (1:100, Santa Cruz, #sc-365782), human anti-Centromere (1:500, Antibodies Incorporated, #15-234), and Alexa flour 594/488-conjugated secondary antibodies (Thermo Fisher Scientific, United States). Rhodamine Phalloidin (1:750, #R415) and PD166285 (#3785) were purchased from Thermo Fisher Scientific and Tocris Bioscience, respectively.

Oocytes at specific stages were fixed in 4% paraformaldehyde for 10 min and permeabilized in phosphate buffered saline (PBS) with 0.1% Triton X-100 for 15 min. Oocytes were then incubated with primary antibodies followed by Alexa Fluor-conjugated 488 and 594 secondary antibodies (Jackson ImmunoResearch). DAPI was used for DNA counterstaining. At least 20 oocytes were examined for each group, unless otherwise stated. The antibodies used in this study were: anti-Zwint-1 (Abcam; 1:50), anti-BubR1 (Abcam; 1:50), anti-Mad2 (Santa Cruz; 1:50), anti-Aurora C kinase (Origene; 1:50), anti-H3S10ph (Millipore; 1:50), anti-acetylated α-tubulin (Sigma; 1:500) and anti-centromere (Antibodies Incorporated; 1:50).
For aneuploidy analysis, oocytes that had been pretreated for 2 hours in 200 μM monastrol (Sigma) were fixed and stained with the indicated antibodies, as described previously45 (link).