Staining of worms with tetramethylrhodamine-phalloidin (catalog # P1951, MilliporeSigma, St. Louis, MO) was performed as described (28 (link), 89 (link)). Samples were observed by epifluorescence using a Nikon Eclipse TE2000 inverted microscope (Nikon Instruments, Tokyo, Japan) with a CFI Plan Fluor ELWD 40 × (NA 0.60) objective. Images were captured by a Hamamatsu ORCA Flash 4.0 LT sCMOS camera (Hamamatsu Photonics, Shizuoka, Japan) and processed by NIS-Elements AR V5.02.01 (Nikon Instruments) and Adobe Photoshop CS3.
To measure fluorescence intensity of AIPL-1::GFP, live worms were mounted and immobilized with 25% Pluronic F-127 (catalog # 2730-50G, Biovision, Milpitas, CA) in M9 buffer containing 0.5 mM levamisole and 0.1% tricaine methanesulfonate. Fluorescence images were captured with the same settings for all samples in NIS-Elements using a Nikon Eclipse TE2000 inverted microscope with a CFI Plan Fluor ELWD 40 × (NA 0.60) objective. Fluorescence intensity of each worm was determined using ImageJ as average fluorescence intensity at 10 randomly selected points within the cytoplasm of the body wall muscle in the head region.
To measure fluorescence intensity of AIPL-1::GFP, live worms were mounted and immobilized with 25% Pluronic F-127 (catalog # 2730-50G, Biovision, Milpitas, CA) in M9 buffer containing 0.5 mM levamisole and 0.1% tricaine methanesulfonate. Fluorescence images were captured with the same settings for all samples in NIS-Elements using a Nikon Eclipse TE2000 inverted microscope with a CFI Plan Fluor ELWD 40 × (NA 0.60) objective. Fluorescence intensity of each worm was determined using ImageJ as average fluorescence intensity at 10 randomly selected points within the cytoplasm of the body wall muscle in the head region.