High-Pressure Freezing and Low-Temperature Processing of Plant Tissue Samples for Electron Microscopy - Plant Cell Morphogenesis: Methods and Protocols - page 148

3.5 Freeze
Substitution and
Embedding in Spurr's
Resin

1. Day 1. Transfer the samples from the liquid nitrogen to 2 %
(w/v) OsO 4 in acetone, contained in cryovials, and store at
−80 °C for 72 h. Use an aluminum block with holes for hold-
ing cryovials. The aluminum block carrying the cryovials can
be placed in a Styrofoam box containing dry ice to maintain a
−80 °C temperature for 3 days.
2. Day 4. Transfer the samples to −20 °C and incubate for 24 h.
3. Day 5. Move the samples to a 4 °C refrigerator and incubate
for an additional 18 h.
4. Day 6. Warm the samples to room temperature for 1 h.
Incubate the samples, which are kept in the same 2 % (w/v)
OsO 4 solution, at 40 °C for 4 h.
5. Remove the osmium solution and replace with acetone; keep
the samples in acetone at 4 °C for 2 h.
6. Cool the samples to −20 °C for 1 h.
7. Remove the acetone and replace with 5 % (w/v) UA in metha-
nol and incubate for 2 h. The UA solution should be cooled
to −20 °C before use.
8. Transfer the samples to acetone cooled to −20 °C and store for
2 h. Warm the samples to room temperature. Tissues are
teased from the planchettes during this time.
9. Transfer the samples to 1 % (v/v) Spurr's resin in acetone and
store for 12 h. Use a resin mix without dimethylaminoethanol
(DMAE).
10. Day 7. Infi ltrate the samples with a graded series (2, 4, 6, 8,
10 % (v/v)) of Spurr's resin diluted in acetone for 2 h each.
11. Day 8. Replace the solution with 25 % (v/v) Spurr's resin
(without DMAE) and infi ltrate for 12 h.
12. Replace the solution with 50 % (v/v) Spurr's resin (without
DMAE) for 12 h.
13. Day 9. Replace the solution with 75 % (v/v) Spurr's resin
(without DMAE) and infi ltrate for 24 h.
14. Day 10. Replace the solution with 100 % (v/v) Spurr's resin
(without DMAE) and infi ltrate for 24 h.
15. Day 11. Replace the solution with fresh 100 % (v/v) Spurr's
resin (without DMAE) and infi ltrate for 24 h.
16. Day 12. Replace the solution with 100 % (v/v) Spurr's resin
(with DMAE) and infi ltrate for 24 h.
17. Day 13. Replace the solution with fresh 100 % (v/v) Spurr's
resin (with DMAE) and infi ltrate for 24 h.
18. Polymerize the resin at 70 °C for 12 h in a vacuum oven.