Biology Reference
In-Depth Information
Fig. 3
Ultrathin sections of adenovirus-infected cells. A549 cells were infected with Ad2 ts1 mutant and pro-
cessed for embedding after 36 hpi. (
a
) Room temperature dehydration and Epon embedding. (
b
) Freeze-
substitution. The insets show details of full (
(filled arrowheads
) and empty (
hollow arrowheads
) capsids imaged
within the cell nucleus. Notice the different contrast obtained by the two methods, particularly for membranes
(
black
in Epon,
white
in freeze-substitution).
C
cytoplasm,
ch
chromatin,
N
nucleus,
ne
nuclear envelope. Scale
bar, 0.5
μ
m
4
Notes
1. Nickel grids must be used for “on grid” immunogold labeling,
instead of copper ones, due to the reactivity of the copper in
saline solutions during prolonged incubations.
2. In the market there are gold particles of different sizes, allowing
the localization of two different epitopes if necessary. Another
alternative is the use of gold conjugated protein A instead of a
secondary antibody (for example Protein A EM-grade 10 nm
Cat#25284 Electron Microscopy Sciences).
3. The presence of glycerol or sucrose in buffers results in dimin-
ished contrast when imaging unstained specimens in frozen-
hydrated conditions, due to the similarity of electron density
between these cryo-protectants and the biological material.
4. Although other electron sources can also be used for cryo-EM
imaging, a Field Emission Gun (FEG) equipped microscope pro-
vides the best quality data to reach subnanometer resolution, due
to the improved coherence of the electron beam generated.
5. The choice of an appropriate substrate is crucial in AFM studies.
The interaction between surface and sample plays a big role in
aspects such as attachment or structural preservation. All the
experiments must be carried out with the same surface since
particles may behave differently due to the interactions with
the surface. For example, adenovirus is unstable on HOPG
(Highly Ordered Pyrolytic Graphite), but it is well attached on
