High-Pressure Freezing and Low-Temperature Processing of Plant Tissue Samples for Electron Microscopy - Plant Cell Morphogenesis: Methods and Protocols - page 149

3.6 Freeze
Substitution and
Embedding in HM20
Resin

1. Day 1. Transfer the samples to 0.1 % GA and 0.25 % UA in
anhydrous acetone and incubate at −80 °C for 72 h in the
AFS2.
2. Day 4. Slowly warm the AFS2 chamber to −45 °C at a rate of
1 °C/h.
3. Day 6. Wash the samples with precooled anhydrous acetone
three times, and start resin embedding by incubating samples in
33 % resin in acetone. Until UV polymerization is completed,
all steps are carried out at −45 °C (from Day 6 to Day 8).

4. Day 7. Increase resin concentration to 66 % and then to 100 %.
Wash samples two times with 100 % resin. Separate samples
from planchettes if they are still attached to the planchettes.
Transfer the samples to fl at-bottomed BEEM capsules, and
place under UV illumination ( see Note 6 ).
5. Day 8. Check whether the HM20 resin has been cured. Allow
the AFS2 chamber to warm to room temperature once the
samples are hardened.

3.7 Sectioning
and Staining

1. Wash slot grids (2 × 1 mm, SPI Supplies, PA) with 1,2-dichlo-
roethane using a sonicator.
2. Clean a glass slide with ethanol or lab detergent and wipe
clean with tissues. Coat the grids with 0.5-1 % (w/v) formvar
solution in 1,2-dichloroethane ( see Note 7 ).
3. For a preliminary observation, cut semi-thin sections (500 nm)
using an ultramicrotome.
4. Stain the sections with 0.1 % (w/v) toluidine blue O in an
aqueous solution containing 1 % (w/v) sodium borate. Check
the sections under an optical microscope to make sure that the
cells are not damaged (Fig. 1b ).
5. Prepare ultrathin sections, post-stain with UA and lead citrate
solutions. Examine the sections under an electron microscope
to confi rm that cells are well preserved.
6. For localization of macromolecules, perform immuno-gold
labeling (as described in refs. [ 26 , 27 ]) prior to post-staining
(Fig. 1c ).

4 Notes

1. We have used an aqueous solution of 0.1 M sucrose for this
purpose. Preservation of tissues after high-pressure freezing is
better when seedlings have been grown with sucrose added to
the culture medium [ 28 ]. Because the germination of onion
seeds was inhibited in the presence of 0.1 M sucrose, seeds