Cultured Rat Hippocampal Neurons

Material and Methods
Rat Hippocampal neurons, cultured on 50 μm thick Aclar (Aclar embedding film, EMS) for 19 days, were frozen in the 100 μm deep side of lecithin coated (detailed protocol Appendix I) type A 3 mm Cu/Au carriers (Leica) and sandwiched with the flat side of lecithin coated type B 3 mm Cu/Au carriers (Leica). No additional filler was used, only cell culture medium with the addition of Hepes buffer pH 7.2 to a final concentration of 25 mM. Samples were frozen in a high-pressure freezer (Leica EM ICE). After freezing sandwiches were opened in liquid nitrogen and the type A carriers were transferred into flow-through capsules (plastic capsules D5 x H15 mm, Leica) to the au­tomated freeze-substitution device (Leica EM AFS2) set at –90° C. Flow-through capsules with samples were placed into Sarstedt tubes with 800 μl Müller’s freeze-substitution medium (Müller et al., 1980), containing 3% (v/v) Glutaraldehyde + 1% (w/v) Osmium tetroxide and 0.3% (w/v) Uranyl Acetate in anhydrous Methanol (detailed protocol Appendix II).

Samples were kept at –90° C for 48 h in this solution and next 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 warmed up from –30° C to +2° C (increment of 10° C/h) and kept at this temperature for 2 h. After this last substitution step samples were washed 2x 5 min with 500 μl anhydrous Methanol, 1x 5 min with 500 μl 50% anhydrous Methanol + 50% anhydrous Acetone and finally 3x 5 min with 500 μl anhydrous Acetone. All washing steps were done in the Leica EM AFS2 at +2° C. Washing steps were carried out by transferring the flow-through capsules with samples to 1.5 ml Eppendorf tubes containing 500 μl precooled washing solution. 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 (detailed protocol Appendix III).

Infiltration with Epon resin/Acetone mixtures was perfor¬med at 4 °C 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)

Infiltration schedule for 100% Epon was performed at room temperature:
pure Epon for 8 h (100% Epon)
pure Epon for 18 h (100% Epon)
pure Epon for 6 h (100% Epon)

Finally, flow-through capsules containing carriers and Aclar discs with neurons were placed in 500 μl Eppendorf tubes containing 250 μl pure Epon, filled up with pure Epon and polymerized for 72 h at 65° C. After polymerization flow-through capsules were taken out of the Eppendorf tubes and opened with a razorblade to take out the sample.The resin around the carrier was trimmed away after which the carrier could be removed from the resin. Next the resin around the Aclar disc was trimmed away after which the Aclar disc could be taken off with forceps leaving the neurons behind in the resin. Approximately 70 nm thick en face 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 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 0.375 nm).