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Array Tomography for serial plastic sections.

Resin options:

1. TAAB Embedding Resin (TER) Kits with BDMA
2. TAAB 812 Resin Kit with BDMA
3. Lowicryl HM20

Workflow:

1. Chemical fixation with half-strength Karnovsky solution (2% paraformaldehyde - 2.5% glutaraldehyde, EM-grade) in sodium cacodylate buffer including 2mM CaCl2 or NaHCa buffer (30mM HPES buffer brought to pH7.4 with NaOH, 100 mM NaCl present to make it roughly isotonic, 2 mM CaCl2 present to preserve cell membranes)
2. Stereo-microscopic anatomy to surely detect an observation target
3. Rapid freeze tissues or cultured cells by Leica EM-HPM100 or Variant Instrument CryoPress (as required)
4. Freeze substitution by Leica EM AFS2 with FSP (as required)
5. Infiltration and resin embedding in epoxy resin by Leica EM-TP
6. Polymerization at 70 °C for a few days by TAAB Embedding Oven with Vacuum Lid or Oven Incubators
7. Semi-thin section and stain with 1% toluidine blue solution
8. Perpendicular trim by Leica EM TRIM2, high speed milling system, prior to ultramicrotomy
9. Serial ultrathin section and ribbon collection by Leica EM ARTOS-3D
10. Mount on the silicon wafers or the EM grids
11. Heavy metal stain with uranyl acetate and citrate lead (as required)
12. Array imaging by TEM, SEM, or confocal fluorescence microscopy
13. 3D-reconstruction and analysis