Alexa fluor 647 affinipure donkey anti human

Oocytes were incubated with 0.5% Triton X-100 for 5 min and then fixed with 4% paraformaldehyde for 30 min. Fixed oocytes were blocked in PBS containing 1% BSA, 0.1% Tween-20 and 0.01% Triton X-100 for 1 h. Oocytes were incubated for 48 h at 4 °C with primary antibodies: rabbit anti-CENPF (Abcam ab5; 1:700), mouse anti-Lamin B1 (Abcam ab8982; 1:400), human anti-centromere (Antibodies Incorporated 15-234-0001; 1:500), mouse anti-DRP1 (Abcam ab56788; 1:200), mouse anti-Tubulin (Abcam; 1:1000) rabbit anti-Tubulin (Abcam ab6046; 1:500) and mouse anti-cMyc (Thermo Fisher R950-25; 1:200). Secondary antibodies used were Rhodamine (TRITC) AffiniPure Donkey Anti-Rabbit (Jackson 711-025-152; 1:750), Alexa Fluor 647 AffiniPure Donkey Anti-Human (Jackson 709-605-149; 1:500), and Alexa Fluor 488 AffiniPure Donkey Anti-Mouse (Jackson 715-545-151; 1:500). DNA was stained with 10 μg/ml Hoechst 33342 (Sigma-Aldrich, St. Louis, MO, USA).

The zona pellucida of oocytes were removed with an acidic M2 medium, and then zona-free oocytes were fixed on the glass slide using chromosome spread solution containing 0.15% Triton X-100, 1% paraformaldehyde and 3 mM dithiothreitol. Immunofluorescent staining was performed after the slides were dried for 12 h. Primary antibodies used were rabbit anti-CENPF (Abcam ab5; 1:50), mouse anti-DRP1 (Abcam ab56788; 1:50), rabbit anti-DRP1 (Abcam ab180769; 1:100), mouse anti-DRP1(BD Biosciences 611112; 1:50), rabbit anti-BUB1 (Abcam ab9000; 1:50), rabbit anti-BUBR1 (Proteintech 115042-AP; 1:100), rabbit anti-APC2 (Proteintech 13559-1-AP; 1:50), rabbit anti-MAD1 (Abcam ab175245; 1:50), rabbit anti-MAD2 (Biolegend 924601; 1:50), human anti-centromere (CREST, Antibodies Incorporated 15-234-0001; 1:100), rabbit anti-REC8 (Proteintech 10793-1-AP; 1:50), rabbit anti-SMC3 (Abcam ab128919; 1:50) and mouse anti-Myc (Thermo Fisher R950-25; 1:100). Secondary antibodies used were Alexa Fluor 647 AffiniPure Donkey Anti-Human (1:50), Rhodamine (TRITC) AffiniPure Donkey Anti-Mouse (Jackson 715-025-150; 1:100), Alexa Fluor 488 AffiniPure Donkey Anti-Mouse (1:100), Alexa Fluor 488 AffiniPure Donkey Anti-Rabbit (Jackson 711-545-152; 1:100), and Rhodamine (TRITC) AffiniPure Donkey Anti-Rabbit (1:100). DNA was stained with 10 μg/ml Hoechst 33342.