Biology Reference
In-Depth Information
2. Place 20-50
l droplets of blocking buffer onto the Parafi lm
(
see
Note 16
;
see
Fig.
2b
for this and following steps).
3. Put grids on top of droplets with section side facing down-
wards and incubate for 30 min at room temperature (
see
Note
17
).
4. Place droplets of the primary antibody solution onto the
Parafi lm.
5. Remove grids from blocking buffer; remove excess liquid by
touching the edge of the grid with fi lter paper (Fig.
2c
) and
place on top of droplets containing the primary antibody.
Incubation time should be 1-2 h at room temperature or up
to 24 h in fridge (
see
Note 18
).
6. Remove grids from droplet with primary antibody solution,
remove excess liquid, and pass grids through a series of at least
four droplets (50
μ
l) of blocking buffer with at least 10 min
incubation each (
see
Note 19
).
7. After removing excess liquid, place grids on top of droplets
containing secondary, gold-conjugated antibody diluted in
blocking buffer and incubate for at least 1 h at room tempera-
ture (or up to 12 h at about 4 °C).
8. Remove excess liquid and pass grids through a series of at least
three droplets of blocking buffer (10 min each) and two drop-
lets of distilled water.
9. Remove grids from droplets and rinse both sides by drop-like
addition of about 5 × 1 ml of distilled water from a Pasteur
pipette. Remove excess liquid as described above and use a
small wedge of fi lter paper to absorb liquid trapped between
prongs of forceps that hold grid (Fig.
2d
).
10. Allow grids to dry on a gridpad with sections facing upwards.
μ
Option 1: After
step 9
use silver enhancement to enlarge the
size of small (2-5 nm) gold particles (
see
Note 20
).
Option 2: After
step 9
stain sections by placing grids on drop-
lets of 2 % aqueous uranyl acetate for 10 min (
see
Note 21
).
4
Notes
1. The conductivity of the water should be
S m
−1
. It is advis-
able to use freshly distilled water not stored for a longer time
in plastic containers.
2. We prefer Trizma pre-set crystals because they need no pH
adjustment. Notice that certain silver chloride pH electrodes
do not give accurate results with Tris buffers. In that case bet-
ter use pH sticks.
≤
1
μ
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