Biology Reference
In-Depth Information
2.4 Chromium
Coating
1. Cressington 308UHR or 328UHR coating unit with sputter
head, chromium target, swinging shutter, “cryopump,” and
fi lm thickness monitor (Cressington Scientifi c Instruments
Ltd., Watford, UK).
2. High purity research grade argon gas (BOC, Guilford, UK).
3. Liquid nitrogen.
4. Clean glass slide (VWR).
Scanning electron microscope, preferably with fi eld emission
source (e.g., Hitachi S-5200 in-lens feiSEM, Hitachi High-Tech,
Tokyo), should be used.
2.5 Observation
by feiSEM
3
Methods
1. Culture cells in dark at 25 °C on a horizontal shaker at
120 rpm.
2. Collect 5 ml of cells by spinning in Falcon tube at 500 ×
g
for
3 min (
see
Note 10
).
3. Remove excess medium.
4. Resuspend cells in enzyme solution (
see
Note 10
).
5. Shake on a horizontal shaker at low rpm (roughly 20-40 rpm)
for 3.5-4.5 h until protoplasts are made (
see
Note 11
).
6. Pellet protoplasts by spinning at 500 ×
g
for 3 min
(
see
Note 10
).
7. Replace the excess enzyme solution with wash buffer.
8. Place on ice (
see
Note 12
).
9. Mix the protoplasts 1:1 with ice-cold lysis buffer.
10. For nuclear isolation, pass the resulting suspension through
the syringe needle 2-4 times (
see
Note 13
).
3.1 Preparing the
Nuclei and Exposing
the Membrane-Bound
Cytoskeleton
3.2 Processing
Samples for feiSEM
1. Prepare microtube chambers as shown in Fig.
3
.
2. Scratch numbers on silicon chips using diamond scribe and
place in glass dish containing acetone.
3. Pick up chips with fi ne tweezers and dry with a tissue to clean.
Chips must be perfectly clean with no sign of smearing on the
surface.
4. Pipette 40
l of poly-
L
-lysine onto each chip and allow to
adhere for 30 min at 25 °C.
5. Rinse the chips in distilled water and allow to dry (
see
Note 14
).
6. Place the chips at the bottom of the microtube chambers
and layer with 200
μ
μ
l of ice-cold fi xative 1 with sucrose. Keep
at 4 °C.
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