Alexa fluor 488 or 633 phalloidin

Animals older than 72 h were anesthetized with MgCl₂ and then killed quickly by adding a small volume (20–30 μl/ml) of 37% formaldehyde directly into the media. They were then fixed in ice cold 3.7% formaldehyde in PBTx [PBS(Phosphate Buffered Saline) + 0.2% Triton X-100] for 30–60 min (when staining for NvLsd1-GFP short fixations yield better staining) or for >60 min or overnight (o/n) (for all other antibodies) at 4 °C. Samples were washed >4 times in PBTx at RT, blocked in Block (3% BSA / 5% Goat serum in PBTx) for > 1 h at RT and incubated in primary antibody diluted in Block o/n at 4 °C. Samples were then washed extensively in PBTx (>5 washes for 2 h or more) at RT, blocked for 1 h at RT in Block and incubated o/n or over the weekend in secondary antibody diluted in Block at 4 °C. If Phalloidin staining was performed, Alexa Fluor™ 488 or 633 Phalloidin (Thermo Fisher Scientific, A12379/ A22284) was added here at 1:50-1:100. Samples were then incubated in Hoechst 33342 (Thermo Fisher Scientific, 62249) at 1:100 in PBTx for 1 h at RT followed by extensive washing in PBTx (>5 washes for 2 h or more). Animals were mounted in ProLong™ Gold Antifade Mountant with DAPI (Thermo Fisher Scientific, P36935) and imaged on a Leica SP5 confocal microscope. Antibodies and dilutions are listed in Supplementary Table 1.

Animals at the planula stage and older were anesthetized with MgCl₂ and then killed quickly by adding a small volume (20–30 μl/ml) of 37% formaldehyde directly into the media. They were then fixed in ice-cold 3.7% formaldehyde in PBTx [PBS(Phosphate Buffered Saline) + 0.2% Triton X-100] for 30–60 min (when staining for NvCoREST short fixations yield better staining) or for > 60 min or o/n (for all other antibodies) at 4 °C. Samples were washed > 4 times in PBTx at RT, blocked in Block (3% BSA/5% Goat serum in PBTx) for > 1 h at RT, and incubated in primary antibody diluted in Block o/n at 4 °C. Samples were then washed extensively in PBTx (> 5 washes for 2 h or more) at RT, blocked for 1 h at RT in Block, and incubated o/n or over the weekend in secondary antibody diluted in Block at 4 °C. If Phalloidin staining was performed, Alexa Fluor™ 488 or 633 Phalloidin (Thermo Fisher Scientific, A12379/A22284) was added here at 1:50–1:100. Samples were then incubated in Hoechst 33,342 (Thermo Fisher Scientific, 62,249) at 1:100 in PBTx for 1 h at RT followed by extensive washing in PBTx (> 5 washes for 2 h or more). Animals were mounted in ProLong™ Gold Antifade Mountant with DAPI (Thermo Fisher Scientific, P36935) and imaged on a Leica SP5 confocal microscope. Antibodies and dilutions are listed in Additional file 1: Table S4.