Biology Reference
In-Depth Information
3.3 Negative
Staining
Immunoelectron
Microscopy
of Purified Viruses
1. Glow discharge collodion/carbon coated Ni grids.
2. Adsorb purified adenovirus particles to the carbon support by
floating the grid on a 3-5 μL drop of the sample for 5 min
(carbon side facing the grid) (
see
Note 9
).
3. Blot by briefly touching the edge of the grid to a piece of
Whatman paper. Do not allow the grid to become completely
d r y.
4. Wash by floating the grid on a drop of TBS for 2 min. Blot as
above.
5. Wash and block by floating on a drop of TBG for 15 min (
see
Note 10
). Blot.
6. Float on a drop of primary antibody at the required dilution
(in TBS/BSA) for 50 min. Several different dilutions should
be tested to optimize the ratio between specific labeling and
background. In parallel, prepare grids labeled with the
positive and negative control antibodies (
see
Fig.
2d, e
). It is
important to keep a high humidity environment by placing
the experiment inside a petri dish containing wet paper all
around the borders.
7. Blot and wash by floating on a drop of TBG, 10 min. Blot.
Repeat twice.
8. Incubate on a drop of secondary antibody conjugated with
10 nm colloidal gold, diluted to 15 % v/v in TBS/BSA for
30 min. Blot.
9. Wash with TBG, 5 min. Blot. Repeat twice.
10. Wash with TBS, 5 min. Blot. Repeat twice.
11. Negative staining with 2 % uranyl acetate for 45 s. Blot and let
dry (
see
steps 9
and
10
in Subheading
3.2
).
1. Glow-discharge Quantifoil grids.
2. Cool down the plunge freezing device using liquid nitrogen.
Once the temperature in the plunging area is below −170 °C,
fill the cryogen reservoir with ethane. Put some cryo-grid
boxes inside the plunging area so they are precooled by the
time the sample is vitrified (
see
Note 11
).
3. Hold one Quantifoil grid with the tweezers. Load the twee-
zers on the plunging device and apply a 2-3 μL drop of adeno-
virus sample to the carbon side. Blot by applying a triangle of
Whatman paper to the carbon face until most of the drop is
removed and only a very thin layer of liquid remains. If using
manual blotting, this step may require extensive hands-on
practice until the desired layer thickness is achieved. Remove
the paper and immediately release the plunging device, to
avoid deleterious evaporation effects.
3.4 Cryo-Electron
Microscopy
