Figure 3.

First midbody remains associated with the fertilized egg following PB1 emission. (A) Fertilized eggs were fixed during meiosis II and stained using Phalloidin::TRITC to label actin and Hoechst to label chromosomes. (i) Confocal images showing actin cap (arrows) during meiosis II in an egg following PB1 removal. (ii) Confocal images showing actin labelling of the midbody (arrows) during meiosis II in an egg following PB1 removal (arrows). (iii) Control egg that displayed PB1 attached to the egg surface. Please note that the midbody is strongly labelled with Phalloidin (arrows). Dotted line indicates surface of the egg. Scale bars = 10 µm. n > 50 for (i) and (ii) where Phalloidin labelled the actin cap (i) and midbody (ii). n > 50 for Phalloidin labelling of outpocket (iii). (B) Fertilized eggs were pipetted during meiosis II to remove PB1, fixed and labelled with anti-phospho aPKC (green), anti-tubulin (red) and DAPI (blue). Left image: overlay showing the rotated second meiotic spindle. The sperm aster is also visible, far left. Scale bar = 30µm. Middle image: inset of boxed region showing that one pole of second meiotic spindle is aligned with PB1 midbody remnant (MR1 arrow). Please note that PB1 was removed by pipetting. Scale bar = 10µm. Right image: Another fertilized egg which had already emitted PB2 showing location of first midbody remnant (MR1, arrows), second midbody remnant (MR2, arrow) and PB2 (arrow). Please note that PB1 was removed by pipetting. n > 50 zygotes where aPKC labelled the midbody remnant. Scale bar = 10µm. Also see Supplementary Movies S4 and S5 of LifeAct labelling.