Muscle tissue was prepared as described in Schwarz et al. (2000) (link). For the preparation of rodent brain tissue the animals were perfused transcardially first with 30 ml of phosphate-buffered saline and then with 40 ml of fixative solution (4% paraformaldehyde in 0.1M PBS [pH 7.4]). The brain tissue was then removed and kept in fixative over night at 4 °C. After being washed twice in PBS, tissue slices (0.2 to 1.5 mm thick) were cut on a vibratome (752 M Vibroslice, Campden Instruments, Leichester, United Kingdom) and kept for 24 h in PBS at 4 °C. Pieces about 1.5 mm in size were then excised and washed three times for 30 min each in cacodylate buffer at pH 7.4.The tissue was postfixed for 2 h in 2% osmium tetroxide/1.5% potassium ferric cyanide in aqueous solution at room temperature. Then the tissue was subjected to a contrast enhancement step by soaking it over night in a solution of 4% uranyl acetate in a 25% methanol/75% water mixture (Stempak and Ward 1964 (link)) at room temperature. After that the tissue was dehydrated in a methanol sequence (25%, 70%, 90%, and 100% for 30 min each) followed by infiltration of the epoxy (Spurr, Epon 812, or Araldite, all from Serva, Heidelberg, Germany) monomer (epoxy/methanol 1:1, for 3 h rotation at room temperature; epoxy/methanol 3:1, overnight at 4°C; pure epoxy, 3 h rotating at room temperature). Polymerization was 48 h at 60 °C for Epon and at 70 °C for Spurr and Araldite. The block face was trimmed to a width of several hundred microns and a length of about 500 μm using either a conventional microtome or a sharp knife. SEM images of the untrimmed block face can be used to select the desired field of view before the final trimming step producing the desired small cutting pyramid.
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