We use Serial-block face SEM for large-volume tissues and cells.
1) Chemically fix tissues/cells with glutaraldehyde & paraformaldehyde mixture.
2) Stain specimen with aqueous osmium tetroxide containing potassium ferrocyanide.
3) Stain with thiocarbohydrazide (TCH) solution.
4) Stain with uranyl acetate solution.
5) Stain with Walton’s lead aspartate.
6) Dehydrate in a series of ethanol dilutions.
7) Infiltrate the specimen into TER-resin.
8) Polymerize at 60°C for 2 days.
9) Confirm the target area by conventional ultra-sectioning.
10) Trim to fine tower.
11) Re-embed in conductive resin.
12) Trim and gold sputtering.
13) Slice and view using Gatan-FEI systems.
14) Perform image processing using FEI AMIRA.
3D reconstruction of Mitochondria in tumor lymphocytes – video: