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Table 2 Post-mortem brain tissue processing for electron microscopy

From: Optimization of ultrastructural preservation of human brain for transmission electron microscopy after long post-mortem intervals

Step Standard fixation and resin embedding Hybrid freezing method
1 Prefixed with 2% paraformaldehyde + 2.5% glutaraldehyde in 0.1 M cacodylate buffer for 24 h at 4 °C
2 Vibratome sections at 150 μm, ROI punched out
3 Post-fixation with OsO4 0.1 M cacodylate buffer for 15 min at RT
4 Washing in 0.1 M cacodylate buffer High pressure freezing with hexadecane as cryoprotectant
5 Dehydration in ascending ethanol series Freeze substitution over 27 h in different substitution cocktails
6 Intermedium propylene oxide Washing in acetone
7 Resin infiltration with ascending resin series in propylene oxide Resin infiltration with ascending resin series in acetone
8 Resin polymerization at 60 °C for 72 h in oven
9 Ultra-thin sectioning at 70 nm and stain grids with lead citrate and platinum blue
10 Electron microscopic examination of ultra-thin sections
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