Chromosome spread
Oocytes were collected and incubated in Tyrode’s solution (Sigma, T1788)
to remove the zona pellucida. After being transferred back to the M2
medium for washing, the zona-free oocytes were gently moved to spread
solution (1% PFA in distilled H2O containing 0.15%
Triton X-100 and 3 mM dithiothreitol, pH 9.2). When the spread solution
droplets were completely dry, the oocytes’ chromosomes were fixed onto
the slide and the following procedure was the same as used for
immunofluorescent staining.
To detect the SAC state, the primary mouse anti-BubR1 antibody (Abcam,
ab54894, 1:300) with a corresponding secondary antibody conjugated with
Alexa Fluor 488 (Thermo Fisher Scientific, A11029, 1:1000) was used.