Immunofluorescence Staining of Worms

The immunofluorescence staining of worms was performed as previously described [43 (link)]. Briefly, the collected worms were permeabilized by flash-freezing in liquid nitrogen. Fixation was carried out by sequential incubation in methanol and acetone at −20°C. The worms were blocked in PBS buffer (Sigma-Aldrich, P4417) containing 5% BSA (Sigma-Aldrich, SLCF3318) and 0.5% Triton X-100 (Sigma-Aldrich, 9036–19-5), then incubated with primary antibodies at 4°C overnight, followed by a series of incubation and washing steps in PBST (0.05% Tween 20 [Sigma-Aldrich, P9416]) buffer. The worms were incubated with secondary antibodies for 1 h, then extensively washed and mounted with anti-fade Fluorescence Mounting Medium (Sigma-Aldrich, F6057). Primary and secondary antibodies were used at the following concentrations: anti-IFB-2 (Developmental Studies Hybridoma Bank [DSHB], 528311; 1:20); anti-DLG-1 (DSHB, 2617529; 1:10); anti-FLAG (Sigma-Aldrich, F7425; 1:50); goat anti-mouse IgG-TRITC (Sigma-Aldrich, T5393; 1:100).