CLEM protocol for meisosome analysis

Sample for CLEM experiments were treated as in Johnson et al., 2015 (link). Briefly, the worms were high-pressure frozen (EMPACT2, Leica) and then freeze-substituted (AFS2, Leica) for 20 hr from –130°C to –45°C in an acetone-based cocktail containing 0.2% uranyl acetate, 0.1% tannic acid, and 5% H₂O. After 2 hr of acetone washes at –45°C, the samples were infiltrated with gradients of HM20 resin over 9 hr, with pure resin for 18 hr at –45°C and the resin was polymerised under UV for 24 hr at –45°C and for 12 hr at 0°C. Then 350 nm semithin sections were processed as described for TEM tomography above. TEM grids were first analysed at by confocal imaging where a bright-field image is overlaid with the fluorescent image, then analysed in TEM at low magnification. Brightfield, GFP confocal, and TEM images were aligned using Amira software. Several positions with two or three GFP spots were chosen to do a high-magnification tomography, as described above, to reveal the meisosomes.