Fig. 8

Localization of COL-99::EGFP::FLAG at NMJs and in other tissues. a Detection of COL-99::EGFP::FLAG expression in the head part of an adult C. elegans. Whole mount immunofluorescence staining was performed with a rabbit anti-GFP antibody and AlexaFluor 488-conjugated anti-rabbit IgG. c Detection of COL-99::EGFP::FLAG in the middle body part. The specimen was prepared by a freeze-crack treatment and a mild PFA fixation. The detection antibody was rabbit anti-GFP. e Staining of methanol/acetone fixed tail (TA) section of a col-99::egfp::flag worm with rabbit anti-GFP (white) and DAPI (blue). The green fluorescence detected with AlexaFluor 488-conjugated secondary antibody was converted to white to enhance the sensitivity. g Double staining of an L1 larva with rabbit anti-GFP (green) and mouse anti-myosin (magenta). The image was converted from a 3D Z-scan and signal was adjusted by Image J software. i Whole-mount immunofluorescent staining of a col-99::egfp::flag adult worm with rabbit anti-GFP (green) and mouse anti-myosin (magenta). k Immunofluorescent staining of a freeze-crack section of worm muscle after mild PFA fixation with rabbit anti-GFP (green) and mouse anti-myosin (magenta). m-o Immunofluorescent staining of a col-99::egfp::flag worm after freeze-crack treatment with rabbit anti-GFP (green in m and o) and α-bungarotoxin (magenta in n and o) showing NMJ localization. o Merged from (m) and (n). q-s Immunofluorescent staining of col-99 embryos with rabbit anti-GFP (green in q and s) and α-bungarotoxin (magenta in r and s). b, d, f, h, j, l, p, and t are negative control staining of unc-119 worms for (a), (c), (e), (g), (i), (k), (o), and (s) respectively. Bars, 5 μm in a-h, k-l and m-p, 10 μm in i-j, and 20 μm in q-t. HE, head; MB, middle body; TA, tail; BM, body muscle; NMJ, neuromuscular junction