C. elegans immunostaining

Embryos from adult hermaphrodites were picked into 10 μl egg buffer on a Poly-L-Lysine coated glass slide (Sigma, St Louis, MO P0425). To release the embryos, a coverslip was placed over the animals and gentle pressure was applied. The slides were subsequently placed on an aluminum plate over dry ice for 1 hr. To crack the embryo’s cuticle and aid its permeabilization, coverslips were quickly snapped off. Slides were fixed in -20°C methanol for 20 min, followed by sequential rehydrations: 80:20, 50:50, and 20:80 methanol to 1x PBS with 0.1% Tween (PBST). After hydration, samples were blocked in 1X PBST with 1% BSA for 1 hr at room temperature and then incubated overnight in primary antibody diluted in PBST at 4°C. Primary antibodies used were anti-tubulin (1:2000, Sigma), and H3 T118ph (1:1000). Samples were then washed with PBST and secondary antibodies were applied for 2 hr at room temperature. Secondary antibodies used were: Alexa Fluor 488 goat anti-mouse IgG and Alexa Fluor 594 goat anti-rabbit (both at 1:1000) (Invitrogen Molecular Probes, Eugene, OR). After incubation with the secondary antibodies the samples were washed with PBST and mounted using ProLong Gold Antifade ProLong with DAPI. Immunofluorescence images were acquired as described below.