Alexa fluor 488 affinipure donkey anti mouse

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.

Myc (to visualize soma targeted receptor) and tdTomato (for M13-CaM transfection confirmation) staining for neuronal cultures was accomplished as follows: individual cultures were rinsed three times in PBS, pH 7.4; slices were blocked in 10% normal donkey serum (Jackson ImmunoResearch, 017-000-121) and 0.1% Triton-X for 30 min and inserted into a shaking incubator set to 23 °C at 120–130 RMP; incubated with a mixture of RFP antibody pre-absorbed (1:1000 in blocking reagent, Rockland antibodies & assays, 600-401-379) and Anti-Myc tag antibody (1:1000 in blocking reagent, Abcam, #ab32) for 90 min in room temperature shaking incubator; rinsed in PBS three times with 5-min incubation periods each time; incubated in Cy3-AffiniPure Donkey Anti-Rabbit IgG (H + L) (1:500, Jackson ImmunoResearch, 711-165-152) and Alexa Fluor 488 AffiniPure Donkey Anti-Mouse (1:500, Jackson ImmunoResearch, 715-545-150) for 30 minutes at room temperature shaking incubator; rinsed with PBS three times with 5-min incubation periods; mounted with DAPI Fluoromount-G (Southern BioTech, 0100-01). All cultures were imaged with a confocal microscope (Zeiss LSM880).