2.2. Phalloidin‐rhodamine technique

For whole‐mount stainings, animals were relaxed in a 1:1 7.14% MgCl₂ × 6H₂O and ASW solution for 15–20 min, fixed in 4% formaldehyde (made from paraformaldehyde) in 1× phosphate buffered saline (PBS) for 1 hr at room temperature (RT) and washed with PBS‐T_x (PBS with 0.1% Triton X‐100, Sigma‐Aldrich) for 1 hr. Specimens were blocked for 1 hr at RT with BSA‐T_x (PBS‐T_x with 1% bovine serum albumin, Carl Roth, Germany) and incubated overnight at 4°C in a rabbit anti‐5HT antibody (Sigma‐Aldrich) diluted 1:2,000 in BSA‐Tx (data from antibody staining not shown here). After being washed with PBS‐T_x for 2 days, specimens were incubated in tetramethylrhodamine‐conjugated phalloidin (P1951; Sigma‐Aldrich) and the secondary fluorescein isothiocyanate‐conjugated (FITC) swine anti‐rabbit antibody (Dako, Denmark), both diluted 1:250 in BSA‐T_x for 1 hr at room temperature in darkness. Subsequently, specimens were rinsed with PBS‐T_x for 3 days at RT and three nights at 4°C in darkness. Finally, they were mounted in Vectashield (Vector Laboratories). In total, 20 adult M. striatum and five adult C. monotrochum were stained.