Steps | Long-term formalin fixed human brains | Fixed-frozen sectioned human brains |
---|---|---|
1 | Postfixed with 4% paraformaldehyde +2.5% glutaraldehyde in 0.1M PB for 2 weeks at 4°C | Postfixed with 4% paraformaldehyde +2.5% glutaraldehyde in 0.1M PB for 14 hours at 4°C |
2 | Vibratome section at 80 μm, store in 0.1M PB | Washed and stored in 0.1M PB |
3 | Osmification in OsO4 0.1M PB for 20 min and washed in 0.1M PB | |
4 | Dehydration in graded ethanol solutions and 100% acetone | |
5 | 1:1 acetone/Araldite for 1 hour at room temperature and in pure Araldite overnight at 4°C | |
6 | Flat embedding on siliconized slides with Araldite | |
7 | Polymeriztion at 60°C for 48 hours in oven | |
8 | Dissection of white matter from embedded sections and glue to blank resin blocks | |
9 | Thin sectioning at 70-80 nm and stain grids with uranyl acetate and lead citrate solutions | |
10 | Examination of ultrathin sections on electron microscope, image processing and quantification |