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-rabbitIgG. 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
a3D Z-scan and signal was adjusted by Image J software. I) Whole-mount immunofluorescent staining of a
col-99::egfp::flag adult worm with rabbitanti-GFP (green) and mouse anti-myosin (magenta). k Immunofluorescent staining of a freeze-crack section of worm muscle after mild PFA fixationwith rabbit anti-GFP (green) and mouse anti-myosin (magenta). m-o Immunofluorescent staining of a
col-99::egfp::flag worm after freezecracktreatment with rabbit anti-GFP (green in m and o) and alpha-bungarotoxin (magenta in n and o) showing NMJ localization. o Mergedfrom (m) and (n). q-s Immunofluorescent staining of
col-99 embryos with rabbit anti-GFP (green in q and s) and alpha -bungarotoxin (magentain 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 um in a-h, k-l and m-p, 10 um in i-j, and 20 um in q-t. HE, head; MB, middle body; TA, tail; BM, body muscle; NMJ, neuromuscular junction