Imaging Plant Nuclei and Membrane-Associated Cytoskeleton by Field Emission Scanning Electron Microscopy - Plant Cell Morphogenesis: Methods and Protocols - page 172

Fig. 3 Diagram of microtube chamber preparation

7. For nuclear isolation, load 10-40 l of sample on the top of

the fi xative 1 in microtube chambers ( see Note 15 ). Spin the
sample onto silicon chips at 2,000 × g for 10 min at 4 °C.
Continue with step 11 .
8. For membrane-bound cytoskeleton, pipette 10-40

μ

l of the
protoplasts from step 9 of Subheading 3.1 onto a silicon chip
( see Note 15 ).
9. Allow 30 min to settle at 25 °C.
10. Fracture the protoplasts attached to the chip by gently touch-
ing a glass coverslip on top of the silicon chip (wet) fracturing
( see Note 16 ).
11. Transfer the chips into fi xative 1 without sucrose and incubate
for 10 min at 4 °C.
12. Transfer the chips into fi xative 2 for 10 min at 25 °C ( see Note 17 ).
13. Rinse the chips in sodium cacodylate for 1 min twice
( see Note 18 ).
14. Transfer chips into a dish containing osmium tetroxide for
10 min ( see Note 19 ).
15. Set out six Petri dishes. Fill one with water and the remainder
with 50, 70, 95, 100, 100 % ethanol, respectively.
16. Transfer chips to each of these for 2 min each ( see Note 20 ).

μ

3.3 Critical
Point Drying

1. Transfer chips to CPD carrier under 100 % dry ethanol.
2. Fill CPD chamber with 100 % ethanol and place specimen car-
rier in chamber.
3. Close lid and start cooling.
4. When cooled to ~10 °C, exchange ethanol for liquid CO 2 with
at least ten changes until all ethanol is replaced.