Biology Reference
In-Depth Information
7. Washing: Place the grid (carbon side down) on the first
washing buffer drop, remove and blot immediately as above.
Repeat the washing/blotting step two more times.
8. Staining: Place the grid on the staining agent drop. Wait for
30 s to 1 min.
9. Pick up the grid, blot, and leave it (facing up) to air-dry on
filter paper in a covered petri dish.
10. Once dried (1-3 h), the grid is ready for observation at the
electron microscope (
see
Fig.
2a, b
) (
see
Note 8
).
Fig. 2
Various techniques to image and characterize purified adenovirus virions. (
a
) General view of a nega-
tively stained electron microscopy preparation. Capsomers appear white, while accumulation of staining agent
around the large viral particle results in a black halo surrounding the virion. (
b
) Detail of a negatively stained
virion where attached fibers are visible (
arrowheads
). The
black rectangle
indicates a loose penton complex
(penton base plus fiber) in the background. Since the height of fibers is much smaller than the virion height on
the substrate, no prominent black halo is observed around them. (
c
) Example of a positively stained viral par-
ticle. Notice that in this kind of image structural detail is lost. Grid areas presenting this kind of stain should be
avoided. (
d
,
e
) Negative staining immunoelectron microscopy. Ad5-derived vectors are probed with an anti-
FLAG monoclonal antibody followed by a goat anti-mouse IgG conjugated with 5 nm colloidal gold particles. In
(
d
), the negative control with Ad5GL vector (structurally wild type) is shown. In (
e
), the antibodies have detected
the presence of the FLAG peptide fused to the C-terminus of polypeptide IX [
38
]. (
f
) Adenovirus imaged by
cryo-EM. The contrast is the opposite as that of negative staining. A loose penton complex is indicated by a
black rectangle
. (
g
) Example of one adenovirus virion imaged by AFM in buffer. A 3D rendering representation
is shown. (
h
) Force-distance curve obtained by indentation on the particle shown in (
g
). (
i
) The same virion
imaged after indentation showing the point in the capsid where breakage occurred. The scale bars represent
100 nm. The AFM panels cover a 300 × 300 nm area
