To determine the structural changes in the cuticle, synchronized L1 larvae [wild-type and npr-8(ok1439) animals] were grown on E. coli OP50 at 20°C until they reached the young adult stage. The animals were then transferred to plates seeded with E. coli OP50 or P. aeruginosa PA14 and cultured at 25°C for 24 hours. Animals were collected and washed with M9 buffer and incubated in the fixation buffer (2.5% glutaraldehyde, 1.0% paraformaldehyde, and 0.1 M sodium cacodylate buffer). For SEM analysis (50), sample suspensions were placed on 0.4-μm Nuclepore filters and dehydrated in graded series of ethanol (10 to 100%) with 10 to 15 min at each grade. The filters were then placed on SEM stubs, sputter-coated with gold:palladium (40:60), and imaged with FEI Quanta 600 FEG SEM. For TEM analysis (50), samples were rinsed with 0.1 M sodium cacodylate buffer and fixed with 1.5% potassium ferrocyanide and 2% osmium tetroxide. The samples were then subjected to T-O-T-O staining followed by dehydration in a graded series of acetone (10 to 100%) with 10 to 15 min at each grade. Samples were infiltrated with Araldite resin, ultrathin-sectioned, and placed on naked gold grids. Images were taken with a FEI Helios NanoLab 650 SEM in STEM mode. For each observation, 20 to 25 cross sections from the midbody region were evaluated, and representative images were presented.

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