Prolong gold antifade mounant

To visualize Actin and TUB^ac, N. vectensis embryos were fixed for 1.5 minutes in 3.7% Formaldehyde, 0.25% Gluteraldehyde in 33% artificial seawater and then in 3.7% Formaldehyde, 0.1% Tween-20 for 1 hour at 4°C. Embryos were blocked in PBST containing 10% donkey serum for 1 hour and subsequently incubated with primary antibodies and Alexa Fluor 555 Phalloidin (Invitrogen) overnight at 4°C. After washing with PBST, the embryos were incubated with secondary antibodies and DAPI (1μg/ml) for 1 hour. Primary and secondary antibodies were diluted in PBST + 2% BSA. Embryos were mounted in ProLong Gold Antifade Mounant (Invitrogen).

S. rosetta colonies were Percoll purified as described above to remove bacteria, placed on poly-L-lysine coated coverslips and incubated for 15 minutes to allow cells to attach. 6% acetone diluted in PEM was applied to the cells for 5 minutes, removed and the cells were subsequently fixed in 4% PFA in PEM for 10 minutes. Cells were blocked with 2% BSA in PEM + 0.1% Triton X-100. To stain for Actin and β-tubulin, embryos were incubated with primary antibodies overnight and secondary antibodies, Alexa Fluor 568 Phalloidin (Invitrogen) and DAPI (1μg/ml) for 1 hour. Coverslips were mounted in ProLong Gold Antifade Mounant (Invitrogen).