Axio imager z1 stand

Worms were anesthetized using 100 mM of sodium azide (NaN₃) and mounted on 5% agarose on glass slides. All images (except Figure 8 and Figure 7—figure supplement 1) were acquired using a Zeiss confocal microscope (LSM880). Several z-stack images (each ~0.4 μm thick) were acquired with the ZEN software. Representative images are shown following orthogonal projection of 2–10 z-stacks. Images shown Figure 8 and Figure 7—figure supplement 1 were taken using an automated fluorescence microscope (Zeiss, AXIO Imager Z1 Stand). Acquisition of several z-stack images (each ~1 μm thick) was performed with the Micro-Manager software (Version 3.1). Representative images are shown following max-projection of 2–10 z-stacks using the maximum intensity projection type. Image reconstruction was performed using ImageJ software (Schneider et al., 2012 (link)).
For quantification of UNC-7::GFP puncta shown in Figure 7C, images were acquired and z-stack were generated as described above. Manual counting of the UNC-7::GFP puncta was performed using the cell counter plug-in of the ImageJ software.
For the quantification of eat-4 and cho-1 expression in AIM for the analysis shown in Figure 5F, images were acquired using a Zeiss confocal microscope (LSM880) and the fluorescence intensity mean was obtained with the ZEN software tool.

Worms were anesthetized using 100 mM of sodium azide (NaN₃) and mounted on 5% agarose on glass slides. Images were taken using an automated fluorescence microscope (Zeiss, AXIO Imager Z1 Stand) or Zeiss confocal microscope (LSM880). Acquisition of several z-stack images (each ~1 µm thick) was performed with the ZEN or Micro-Manager software (Version 3.1) (Edelstein et al., 2010 (link)). Representative images are shown following max-projection of 2–10 µm Z-stacks using the maximum intensity projection type. Image reconstruction was performed using Image J software (Schneider et al., 2012 (link)).

Animals of the respective genotypes were grown at 20 °C on nematode growth media (NGM). We followed an immunocytochemical staining procedure described previously^{64 (link)}. In brief, worms were prepared for staining following the freeze-crack procedure and subsequently fixed in ice-cold acetone (5 min) and ice-cold methanol (5 min). Worms were transferred using a Pasteur pipette from slides to a 50 ml conical tube that contained 40 ml 1 × PBS. Following brief centrifugation (2 min, 1800 × g), worms were pelleted and 1 × PBS was removed. Next, worms were incubated with 300 μl of blocking solution (1 × PBS, 0.2% Gelatin, 0.25% Triton X100) for 30 min at room temperature (rolling agitation). Following removal of the blocking solution, worms were incubated overnight with a mouse anti-poly-GA antibody (1:25 in PGT solution, EMD Millipore). Next, the primary antibody solution was removed and worms were washed five times with washing solution (1 × PBS, 0.25% Triton X100). Worms were incubated with an Alexa Fluor 594 goat anti-mouse IgG secondary antibody (1:1000 in PGT solution, A-11020, Molecular Probes) for 3 h at room temperature. Following 5 washes, worms were mounted on a glass slide and examined with an automated fluorescence microscope (Zeiss, AXIO Imager Z1 Stand).