Biology Reference
In-Depth Information
2.4 Cryo-Electron
Microscopy
1. Holey carbon covered grids (e.g., Quantifoil R 2/4).
2. Manual or automated plunge-freezing device (e.g., Leica CPC,
FEI Vitrobot) [
7
].
3. High precision grade tweezers compatible with plunge-
freezing device to be used (e.g., Dumont #L5 Medical Forceps
with Slide for Leica CPC).
4. Liquid nitrogen. Caution: extremely cold. Wear safety eyewear.
Use in well-ventilated areas.
5. Ethane gas. Caution: highly flammable. Do not use in the pres-
ence of an open flame. Wear safety eyewear. Dispose by allowing
to evaporate in fume hood. Use in well-ventilated areas.
6. Whatman Grade Nº 1 Filter paper, 90 mm diameter.
7. Purified virus sample at 5 × 10
12
vp/mL in sucrose or glycerol
free buffer (
see
Note 3
).
8. Cryo-grid boxes for storage and transfer under liquid nitrogen
(e.g., Ted Pella cat# 160-40).
9. Liquid nitrogen tank for storage of vitrified samples (AirLiquide
GT35 or similar).
10. Access to a transmission electron microscope (FEI Tecnai G20
or similar) equipped with cryo-holder (e.g., GATAN 626) and
low dose image acquisition system (
see
Note 4
).
1. Atomic force microscope equipped with operating and image
processing software WSxM [
24
] (e.g., Nanotec Dulcinea,
Nanotec, Madrid, Spain).
2. Standard Silicon Nitride Rectangular RC800 (20 × 200 μm,
0.05 N/m and 40 × 200 μm, 0.1 N/m) and Biolever RC150
(30 × 60 μm, 0.03 N/m) cantilevers (Olympus, Japan).
3. Purified adenovirus sample at a concentration of ~5 × 10
12
vp/
mL stored in HBS buffer (20 mM HEPES, 150 mM NaCl,
pH 7.8) in single-use (5 μl) aliquots at −70 °C.
4. Stock solution of NiCl
2
in HBS buffer at the required concen-
tration to give 5 mM Ni
2+
in the final sample.
5. Flat, clean surface for sample attachment and support (e.g.,
Muscovite mica V-1 quality) (
see
Note 5
).
2.5 Atomic Force
Microscopy
1. Adenovirus-infected and mock-infected cells at the appropriate
post-infection time(s), grown in monolayer in 10 cm diameter
culture plates. Usually one plate is enough to obtain three
blocks of sample for sectioning.
2. 0.4 M HEPES buffer, pH 7.2.
2.6 Electron
Microscopy
of Infected Cells
2.6.1 Fixation,
Dehydration, and
Embedding in Epoxy
Resins for Morphological
Studies
3. PBS: 8.01 g/L NaCl, 0.20 g/L KCl, 1.78 g/L Na
2
HPO
4
∙2H
2
O,
0.27 g/L KH
2
PO
4
, pH 7.4.
