Biology Reference
In-Depth Information
DTT (dithiothreitol, Sigma-Aldrich), and 0.2 mM PMSF
(phenylmethylsulfonyl fl uoride, Sigma-Aldrich). Make fresh as
required (
see
Note 3
).
10. 0.5 × 16 mm syringe needle (Terumo Europe, Leuven,
Belgium).
11. 5 ml syringe (VWR International, Lutterworth, UK).
12. Membrane fi lter, pore size 0.2
μ
m (VWR).
1. 5 mm × 5 mm silicon chips (Agar Scientifi c Ltd., Stansted, UK).
2. Acetone.
3. Poly-
L
-lysine: 1 mg/ml in water (Sigma-Aldrich) stored at
−20 °C.
4. Distilled water.
5. Microtubes for making microtube chambers.
6. Microtube cutter (Science Services, München, Germany).
7. Scissors.
8. Diamond scribe for labelling silicon mounts (Agar Scientifi c
Ltd., Stansted, UK).
9. PME buffer: 100 mM PIPES, pH 6.4, 5 mM MgCl
2
, 10 mM
EGTA (Sigma-Aldrich), and 0.2 M sucrose (VWR
International;
see
Note 4
).
10. Fixative 1: 3 % [w/v] paraformaldehyde (Agar Scientifi c Ltd.)
and 0.2 % [w/v] glutaraldehyde (Sigma-Aldrich) in PME buf-
fer. Make fresh as required (
see
Notes 5
and
6
).
11. Fixative 1 without sucrose. Make fresh as required (
see
Note 5
).
12. Fixative 2: 2 % [w/v] glutaraldehyde and 0.2 % [w/v] tannic
acid in PME buffer (all chemicals obtained from Sigma-
Aldrich). Make fresh as required.
13. Cover slides.
14. Dumont tweezers 5/45 (Agar Scientifi c Ltd.).
15. 35 mm Petri dishes (Jencons-PLS, East Grinstead, UK).
16. 0.1 M sodium cacodylate pH 7.4 (
see
Note 7
). Store at room
temperature up to 1 month.
17. 0.1 % OsO
4
(Agar Scientifi c Ltd.) in 0.1 M sodium cacodylate
pH 7.4. Store at room temperature up to 1 month (
see
Note 8
).
18. 50, 70, 95, 100 % ethanol (
see
Note 9
).
2.2 Processing
Samples for feiSEM
1. Bal-Tec CPD030 (now Leica Microsystems GmbH, Wetzlar,
Germany) (other makes are also suitable) with exit gas fl ow
meter and connected to high purity CO
2
cylinder with liquid
withdrawal.
2. 100 % ethanol (
see
Note 9
).
2.3 Critical
Point Drying
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