Biology Reference
In-Depth Information
3. Liquid nitrogen.
4. Onion seeds.
5. BY-2 suspension-cultured cells (liquid culture).
6. Fresh ear of corn.
7. Cryoprotectant: 0.1-0.15 M sucrose solution in distilled water
or 20 % dextran (molecular weight: ~40,000, Cat no. 31398
from Sigma) in BY-2 cell culture medium.
8. Vacuum fi ltration funnel and 30
m pore size nylon fi lter.
9. Biopsy punch (diameter: 2 mm; Miltex, Plainsboro, NJ).
μ
2.2 Freeze
Substitution
1. 2 % OsO
4
in acetone: dissolve 1 g of OsO
4
in 50 ml anhydrous
acetone. Aliquot into cryovials (approximately 1 ml per vial).
Freeze in liquid nitrogen and store frozen until use.
2. 0.1 % glutaraldehyde (GA) and 0.25 % UA in acetone: prepare
25 % uranyl acetate in anhydrous methanol. Dilute a 10 % GA
solution in acetone (Electron Microscopy Sciences, Hatfi eld,
PA) and the 25 % methanolic UA solution with acetone to
fi nal concentrations of 0.1 % GA and 0.25 % UA. Aliquot into
cryovials, freeze in liquid nitrogen, and store frozen until use.
3. AFS2 automatic freeze substitution machine (Leica
Microsystems, Buffalo Grove, IL).
2.3 Embedding
1. Embed-812 kit or low viscosity Spurr's kit (Electron
Microscopy Sciences, Hatfi eld, PA).
2. Lowicryl HM20 resin kit (Electron Microscopy Sciences,
Hatfi eld, PA).
3. Flat-bottomed BEEM capsules (Electron Microscopy Sciences,
Hatfi eld, PA).
1. UC-7 (Leica Microsystems, Buffalo Grove, IL) or a similar
ultramicrotome.
2. Formvar-coated EM grids (copper or nickel slot grids,
1 mm × 2 mm slot size).
3. Diamond knife.
4. 2 % (w/v) UA in 60 % ethanol or 2 % aqueous UA and
Reynolds' lead citrate solution.
2.4 Ultramicrotomy
and Post-staining
3
Methods
3.1 Growing Onion
Seedlings
1. Sow onion (
Allium cepa L
. cv. Highgold Nigou) seeds on a
piece of fi lter paper (No. 1) wetted with 0.05 M sucrose in
distilled water in a plastic Petri dish (
see
Note 1
). Use parafi lm
to seal the lid of the dish to avoid drying of the fi lter paper.
Cover the container with a sheet of aluminum foil to allow the
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