Biology Reference
In-Depth Information
5.
Break the carbon film into sections of approximately five grids per side. Cut
strips of newspaper slightly larger than the raft of grids (
see
Note 8
). Gently lay
the paper onto the grids and carbon film. When the water has completely soaked
through the newspaper, lift the paper and grids onto a piece of filter paper to dry.
Store carbon films on dry filter paper in a Petri dish.
3.4. Preparation of Unstained Sample Grids
1.
Prepare a range of sample dilutions (e.g., undiluted, one-third, one-sixth, and
one-ninth) in fibrillar collagen extraction buffer or column buffer as appropriate.
2.
Fold a circular filter paper into quarters. Using tweezers, with a clamping ring,
grip the centre of the carbon-coated microscope grid. Pipet 5-6
L of the sample
onto the centre of the grid. Allow the sample to adsorb for 30 s (
see
Note 9
).
µ
3.
Gently wick off excess sample against the folded side of the filter paper. Wash
the grid with three successive drops of water then drain excess water from below
the grid.
4.
Allow the grid to air-dry for 5 min prior to examination in the microscope (
see
Note 10
).
3.5. Rotary Shadowing
This protocol for rotary shadowing is based on the mica sandwich tech-
nique originally described by Mould
(8)
and subsequently adapted
(9
,
10)
.
Alternatively, unstained grids may be prepared as described in protocol
Sub-
heading 3.4.
, examined by STEM and subsequently shadowed as described in
steps 8
onward.
1.
Prepare a range of sample dilutions (e.g., undiluted, 1/3, 1/6 and 1/9) in 0.2
M
ammonium acetate.
2.
Cleave a 2 cm
×
2 cm mica sheet (
see
Note
6).
3.
Submerge a freeze-drying table in a liquid nitrogen bath to cool. Replenish the
liquid nitrogen frequently maintaining a layer of nitrogen over the table.
4.
Pipet 5
L of the sample onto the inner surface of one mica sheet. Lay the second
mica sheet onto the first (inner surface to inner surface) overlapping the edge by
1-2 mm.
µ
5.
After 10 min at room temperature, split the mica sandwich under 0.2
M
ammo-
nium acetate. Keeping the mica pieces immersed in the buffer, wash by gentle
agitation for one min before reclosing the sandwich with a 1-2-mm overlap.
6.
Plunge the closed mica sandwich into a liquid nitrogen bath. Using a pair of
forceps in each hand, grip the opposing mica sheets and split open. Transfer the
mica sheets onto the submerged freezing table, inner surfaces facing upwards.
7.
Follow the manufacturers instructions to freeze-dry the mica sheets on the cold table.
Freeze-dried mica sheets may be stored in a clean/dry environment indefinitely.
8.
Cut a length of tungsten wire to a length appropriate to the dimensions of the
holding jig. Cut 8 cm of platinum wire and wind in a spiral around the tungsten.
Squeeze the wound platinum to cover a length of 1 cm. Place the tungsten/plati-
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