Imaging Plant Nuclei and Membrane-Associated Cytoskeleton by Field Emission Scanning Electron Microscopy - Plant Cell Morphogenesis: Methods and Protocols - page 174

−20 °C up to 6 months. PMSF is toxic and should be handled
very carefully in a fume hood.
4. PME buffer can be stored at −20 °C up to 1 month.
5. Caution when working with paraformaldehyde.
Paraformaldehyde is toxic by ingestion or inhalation. It can
cause skin corrosion and serious eye damage. Handle as a car-
cinogen, work in a fume hood, and wear gloves.
6. Dissolve PFA by heating the solution in a water bath at 60 °C
maximally. It is important to keep the temperature stable as
dissolving at higher temperatures the fi xative loses its fi xing
properties whereas at lower temperatures it would take too
long to dissolve.
7. Caution when working with sodium cacodylate. May be fatal if
swallowed. Known carcinogen in humans. Harmful if inhaled,
may be harmful by skin contact. Long-term exposure may lead
to kidney and liver damage. Eye and skin irritant. Use gloves,
safety glasses, and good ventilation. Handle as a carcinogen.

8. Caution: osmium tetroxide is volatile, toxic by inhalation and
by skin contact. Work in a fume hood and wear gloves.
9. 100 % ethanol should be stored with Molecular Sieve to ensure
that it is dry.
10. Be extremely cautious and gentle when working with cells and
protoplast, use pipette tips with cut tip, and resuspend cells by
gently tapping the Falcon tube.
11. In the meantime you can prepare microtube chambers and
silicon chips (Subheading 3.2 , Steps 1 - 6 ).
12. All subsequent steps are performed on ice.
13. Use gentle pressure when passing the suspension through the
syringe needle.
14. Silicon chips can be stored at room temperature up to 1
month.
15. Experience is needed to estimate the amount. The volume of
the loaded sample can be adjusted according to the cellular
density. As a rough guide, if the suspension has a nearly milky
color, use 10 l.

16. No pressure is required; just place the cover slide on the chip
and remove immediately.
17. This step can be performed also at 4 °C for several hours or
overnight; thus, the timing can be adapted as needed.
18. We recommend transferring chips between dishes containing
the required solution rather than changing solution in one dish.
19. Ten minutes is suffi cient for thin samples such as nuclei and
subcellular components, but longer times (30-60 min) are
required for bulkier samples such as pieces of tissue.

μ l or less; if lightly cloudy, use 20-40 μ