Paraformaldehyde (pfa)

Immunofluorescence labeling was carried out using standard protocols (cf.⁴⁸ ). Fixation was performed without any washing steps in order to avoid cell retraction. Three different fixation methods were used: (i) For methanol acetone fixation, cells were fixed for 2 min in methanol at −20 °C and immediately permeabilized in acetone at −20 °C for 20 s. (ii) For paraformaledehyde (PFA)-acetone fixation, cells were first fixed for 10 min at room temperature in 4% (w/v) PFA (Merck) in PBS and were then permeabilized in acetone at −20 °C for 30 s. (iii) For PFA-Triton X fixation, cells were first fixed for 10 min at room temperature in 4% (w/v) PFA in PBS, and were then permeabilized in 2% Triton X-100 (Sigma) in PBS solution for 10 min at room temperature.
For staining, fixed cells were passivated for 20 min at room temperature in a solution of 5% (w/v) standard grade bovine serum albumin fraction V (BSA; Serva) in PBS. Incubation with primary antibodies in a solution of 1% (w/v) BSA in PBS was performed for 90 min at room temperature and followed by three washing steps with PBS. Incubation with the secondary antibodies suspended in PBS with 1% BSA was performed for 35 min at room temperature and followed by three washing steps. Mounting reagent was Mowiol (Carl Roth).
A complete list of antibodies used for immunofluorescence is provided in SI Table 1.

Prior to processing flukes for confocal microscopy, worms were paralysed by incubation in 7.14% MgCl₂ for 1 min at RT. Juveniles cultured for up to 3 weeks post-excystment were free-fixed with rotation in 4% paraformaldehyde (PFA: 4% PFA (Sigma Aldrich) in PBS, pH 7.4) for 4 h at room temperature (RT, 21–25°C). Larger worms (i.e. > 4 weeks after excystment) were flat fixed in 4% paraformaldehyde between microscope slides for 2 h and then free-fixed as described above for a further 2 h, all at RT. Fixed worms were subsequently subjected to 3x 15 min washes in PBSTx (PBS containing 0.5% Triton X-100 (Sigma-Aldrich)) at RT with a final overnight wash at 4°C. Juveniles were then incubated in tetramethylrhodamine isothiocyanate (TRITC)-conjugated phalloidin (Sigma-Aldrich; 200 ng/μl in Antibody Diluent (AbD: PBS containing 0.1% bovine serum albumin (Sigma-Aldrich) and 0.1% Triton X-100 (Sigma-Aldrich)) for 4 h in the dark at RT, before 3x 15 min washes in AbD at RT. Worms were mounted on slides in 8 μl of Vectashield (Vector labs) and viewed under a Leica AOBS SP5 confocal scanning laser microscope.