Samples were kept at –90 °C for 48 h in this solution, next washed 4x 30 min at –90 °C with 500 μl precooled 2% (w/v) Osmium tetroxide in anhydrous Acetone and finally left for 46 hat –90 °C in 2.0 ml Sarstedt tubes containing 500 μl precooled 2% (w/v) Osmium tetroxide in anhydrous Acetone. Washing steps were carried out by transferring the flow-through capsules with samples to 2.0 ml Sarstedt tubes containing 500 μl precooled 2% (w/v) Osmium tetroxide in anhydrous Acetone.
Next samples were gradually warmed from –90 °C to –60 °C (increment of 2 °C/h); kept at –60 °C for 8 h before warmed up to –30 °C (increment of 2 °C/h). At –30 °C samples were kept for 8 h and finally were placed on ice for 1 h followed by 1 h at room temperature. Samples were washed 3x 5 min with anhydrous Acetone by transferring the flow-through capsules with samples to 1.5 ml Eppendorf tubes containing 500 μl anhydrous Acetone. A volume of 800 μl freeze-substitution medium was chosen to have enough capacity to replace the water in the sample for Acetone. For all next steps a volume of 500 μl was used to prevent loss of the sample out of the flow-through capsule by overflow.
After the last wash with anhydrous Acetone samples were infiltrated with Epon resin (see detailed protocol****). Infiltration with Epon resin/ Acetone mixtures was perfor¬med at room temperature by the following steps:
Epon : Acetone = 1 : 2 for 2 h (33% (v/v) Epon)
Epon : Acetone = 1 : 1 for 18 h (50% (v/v) Epon)
Epon : Acetone = 2 : 1 for 8 h (66% (v/v) Epon)
Epon : Acetone = 3 : 1 for 18 h (75% (v/v) Epon)
pure Epon for 8 h (100% Epon)
pure Epon for 18 h (100% Epon)
pure Epon for 6 h (100% Epon)
Finally, individual C. elegans were taken out of the type A carriers and placed separately in the cavities of an embedding mould (Aclar scientific). The cavities were filled with pure EPON and polymerized for 72 h at 65 °C. Approximately 70 nm thick sections were cut on an ultramicrotome (Ultracut UCT, Leica) and collected on Carbon coated-Formvar-50 mesh hexagonal copper grids. The sections were contrasted 6 min with 7% (w/v) Uranyl Acetate in 70% Methanol and 2 min with Reynolds Lead Citrate.
Micrographs were acquired on a Tecnai 12 electron microscope (FEI) operated at 100 kV and spotsize 3. Electron micrographs (Binning1, 2048x2048 pixels) were collected with a TIETZ camera (TIETZ TVIPS TemCam F214) at 15,000x magnification (pixel size 0.375nm).
Lecithin coating of carriers:
Prepare a solution of 2% (w/v) Lecithin (L-α-Phosphatidylcholine from egg yolk, Fluka-61755) in Chloroform. Dip the type A carriers one by one in the Lecithin solution and lay the carriers with the 100 μm deep side up on filter paper. Dip also the type B carriers in the Lecithin solution and lay the carriers with the flat side up on filter paper. The carriers will be covered with a thin layer of Lecithin after the Chloroform has evaporated.
C.elegans extracellular filler:
For 2 ml: 20 mg Agarose, type IX (Ultra-low gellingpoint Agarose, Sigma-Aldrich A-5030) in 1.6 ml bacteria medium, dissolve at 60 °C. Let the solution cool down to 37 °C, add 400 μl 10% (w/v) BSA (Bovine Serum Albumin, fraction V, Sigma-Aldrich A-9647) in milliQ water and mix gently.
Procedure described in “One Protocol Fits All” Matthia Karreman et al.: Lights will guide you, Sample Preparation and Applications of integrated Laser and Electron Microscopy, published by Uitgeverij BOXPress, ‘s-Hertogenbosch, The Netherlands, ISBN: 978-90-889-1562-8
For approximately 50 ml pure Epon add together:
1. 21.5 gram Epoxy embedding medium (Fluka)
2. 25.0 gram Araldite M Hardener 964 (Fluka)
3. 7.0 gram Epoxy embedding medium hardener MNA (Fluka)
Mix well for at least 10 min and add:
4. 0.75 ml Epoxy embedding medium accelerator DMP 30 (Fluka)
Mix well for at least another 10 min