Affinipure donkey anti rabbit

Citrated blood freshly collected from HIT patients and healthy donors was diluted in PBS and stained with anti-CD15 (Alexa 647, BD 562369), anti-CD16 (APC-Cy7, BD 557758), anti-CD41a (V450, BD 561425), anti-citrullinated histone H3 (ab5103), anti-MPO (PE, BD 341642) and Affinipure donkey anti-rabbit (Alexa 488, Jackson ImmunoResearch Laboratories 711545152) antibodies. Samples were fixed with paraformaldehyde and analysed by flow cytometry (CantoII, BD Bioscience). Citrated blood from healthy donors was also treated in vitro with HIT or control antibodies, labelled and analysed as described above. Fluorescent counting beads (Invitrogen C36950) were used to derive absolute cell numbers by flow cytometry following the manufacturer’s instructions. Platelet counts from animal experiments were determined by the number of events acquired in 60 s. LDG and monocytes from human and mouse experiments isolated using Histopaque separation media (Sigma)⁴⁹ were identified using anti-CD15 (Alexa 647, BD 562369), anti-CD16 (APC-Cy7, BD 557758, anti-CD14 (V500, BD 561391), anti-Ly6G (V450, BD560603) and anti-CD11b (APC, BioLegend 101212).

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.