Freeze-Fracture Replication of Pyramidal Cells

Frozen samples (90 μm thick slices frozen by Leica EM HPM100) were inserted into a double replica table and then fractured into two pieces at –130°C (after insertion of the tissue into Leica BAF 060 the samples should be left in the chamber for 20 min to reach the –130°C). Fractured faces were replicated by (i) deposition of carbon (5 nm) at 45° angle with sample rotation, (ii) platinum/carbon (2 nm) at 60° angle without sample rotation, (iii) carbon (15-20 nm) at 45° angle with sample rotation. Replicas are transferred into 2.5% SDS solution and samples are incubated at +80°C for 18 hours with continuous shaking.