Materials and Reagents

Ethanol
Acetone
Na₂HPO₄
Gelatin capsules (Electron Microscopy Sciences, catalog number: 71012 )
Glutaraldehyde (Fluka, catalog number: 49627 )
Paraformaldehyde
OsO₄
Catalyst
Osmium (Fluka, catalog number: 75633 )
Fixative solution (see Recipes)
Post-fixative solution (see Recipes)
NaPO₄buffer (see Recipes)
Resin (LR white) (Fluka, catalog number: 62662 ) (see Recipes)

Equipment

Shaker
Incubator with temperature control
Light or electron microscopy
Vacuum
Glass blade or jeweler saw (Electron Microscopy Sciences, catalog number: 71012 )

Procedure

Fixation: Cut and fix plant tissues in fixative solution for 2 h, then vacuum for 15 min without shaking. If the tissues did not sink, re-infiltrated for another 10 min. The tissues can be stored at 4 °C over night.
Rinse: Rinse the entire samples in 0.5 M NaPO₄ buffer, and then wash tissues in 0.5 M NaPO₄ buffer for 3 times; 15 min each at 4 °C. Add enough NaPO₄ buffer to cover the entire tissues.
Post-fix: Transfer samples to 1% Osmium (OsO₄) in 0.5 M NaPO₄ buffer for 1 h in the dark. The samples should be immersed in the post-fixative solution. The tissues can be kept for another hour in post-fixative solution until they all turned black.
Wash without changing container: 3 times in ddH₂O; 10 min each at 4 °C on a rotating shaker.
Dehydration without shaking: Dehydrate the tissues in a graded ethanol series as below:
12.5% ethanol, 10 min
25% ethanol, 10 min
35% ethanol, 10 min
50% ethanol, 10 min
70% ethanol, 10 min
80% ethanol, 10 min
95% ethanol, 10 min
100% ethanol, 10 min (twice)
100% acetone, 10min (twice)
Infiltration: To damp the old solution and exchange the 100% ethanol with acetone-resin mixture as following steps:
Acetone: resin (1:1) 1 h
Acetone: resin (1:2) 1/2 h
100% resin 1 h
100% resin over night on shaker
Polymerize: Put samples in gelatin capsules filled with 100% fresh resin, leave samples at 50 °C for 60 h. Once the resin was solidified, the capsules can be kept at 4 °C for a few weeks.
Blocks can be trimmed and cut with glass blade or jewelers saw depending on what shapes were required for light or electron microscopy. To position the blocks depend on the dimension (cross, transverse, longitudinal) section layout was required. The sections were picked up and floated out on 30% acetone on a warmer plate (42 °C) until dry.

Recipes

Fixative solution (make fresh with ddH₂O)
3% Glutaraldehyde (10% stock)
1.5% Paraformaldehyde (16% stock)
1.6% 1 M NaPO₄
Post-fixative solution (make fresh)
OsO₄ (4% stock) 2 ml
1 M NaPO₄ 4 ml
H₂O 2 ml
1 M NaPO₄ buffer (pH 6.8)
NaH₂PO₄.H₂O 137.99 g/L
Na₂HPO₄ 141.97 g/L
Resin (LR white)
Add 9.9 g catalyst to 500 ml LR white, shaken thoroughly.

Acknowledgments

This protocol is adapted from Alpers and Beckstead (1985) and Massover (2011).

References

Alpers, C. E. and Beckstead, J. H. (1985). Enzyme histochemistry in plastic-embedded sections of normal and diseased kidneys. Am J Clin Pathol 83(5): 605-612.
Massover, W. H. (2011). New and unconventional approaches for advancing resolution in biological transmission electron microscopy by improving macromolecular specimen preparation and preservation. Micron 42(2): 141-151.