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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