Op50 e coli

HPF and FS were optimized based on previous work in both C. elegans and Drosophila (Rostaing et al., 2004 (link); Fouquet et al., 2009 (link); Gracheva et al., 2010 (link); Stigloher et al., 2011 (link); McDonald et al., 2012 (link)). Late second-instar larvae were placed in the 200-µm cavity of aluminum specimen carriers (Type A, with the flat side of Type B carriers used as a lid; Technotrade International) filled with a mixture of 10% BSA (Sigma-Aldrich) and OP50 E. coli in PBS. Specimen carriers were pretreated with 1-Hexadecene (Sigma-Aldrich) to adequately seal the freezing chamber. HPF was performed with a Bal-Tec HPM-010 apparatus at a freezing speed >20,000 K/s and pressure >2,000 bar. FS was performed in a Leica EM AFS1, where samples were incubated in 0.5% glutaraldehyde, 0.1% tannic acid, and 1% H₂O in anhydrous acetone for 98 h at −90°C, rinsed with 1% H₂O in acetone for 2 h at −90°C followed by incubation in 1% OsO4 and 1% H₂O in acetone for 7 h at −90°C. Samples were warmed to −20°C over 14 h (5°C/h), then incubated at −20°C for 16 h. Temperature was increased to 4°C over 2.4 h (10°/h). Finally, samples were rinsed with 1% H₂O in acetone for 2 h, transferred to RT, and embedded in epon using standard procedures.