Diamond knife

Mouse hippocampus were isolated and fixed in 2.5% glutaraldehyde (Sigma-Aldrich, G6257) at 4°C overnight. The samples were washed in 0.1M sodium cacodylate buffer and post-fixed in 1% OsO₄ at R.T. for 2 hours. The tissues were dehydrated in ethanol with increasing concentrations, followed by infiltration in epoxy resin/propylene oxide (1:1) at 37°C overnight. Tissues were embedded in epoxy resin at 60°C overnight. Ultra-thin sections were cut with a diamond knife (Diatome) and placed onto copper grids followed by staining with silver citrate. Images were captured by a Philips CM100 transmission electron microscope.

Concentrated cells were high pressure frozen (HPF Compact 02; Wohlwend, CH), freeze substituted (in acetone containing 0.25% osmium tetroxide, 0.2% uranyl acetate, and 5% water) (AFS2; Leica, Wetzlar, Germany) and embedded in Epon 812 substitute resin. Freeze-substitution was done as follows: −90 °C for 20 h, from −90 °C to −60 °C for 1 h, −60 °C for 8 h, −60 °C to −30 °C for 1 h, −30 °C for 8 h, and −30 °C to 0 °C for 1 h. At 0 °C, samples were washed three times with acetone before a 1:1 mixture of Epon 812 substitute resin (Fluka Chemical, Buchs, Switzerland) and acetone was applied at room temperature. After 2 h the 1:1 mixture was substituted with pure resin overnight. After another substitution with fresh Epon, samples were polymerized at 60 °C for 2 days. Using an ultramicrotome (UC7; Leica) and a diamond knife (Diatome, Biel, Switzerland) 50 nm sections were cut from the Epon blocks and transferred to 100-mesh copper grids coated with pioloform. For additional contrast, sections were poststained with 2% uranyl acetate for 20–30 min and subsequently with lead citrate for another 1–2 min. Sections were finally analyzed and imaged using a JEM-2100 transmission electron microscope (JEOL, Tokyo, Japan) equipped with a 2k × 2k F214 fast-scan CCD camera (TVIPS, Gauting, Germany).