Biology Reference
In-Depth Information
3. Lateral distances appear larger due to tip-sample dilation or to
drift during acquisition.
4. Nanoindentation curves can be depicted in force vs. deformation
plots by subtracting the curve of the cantilever deflection on
substrate (
point 12
above) from the curve of the deformation
of the cantilever-virus system. The nanoindentation curve pro-
vides information about the stiffness and breaking force of the
viral capsid.
3.6 Electron
Microscopy
of Infected Cells
1. Prepare fixative (2 % glutaraldehyde, 1 % tannic acid in 0.4 M
HEPES, pH 7.2). Dissolve 0.5 g of tannic acid in 46 mL of
400 mM HEPES, pH 7.2. Filter the solution with a 0.22 μm
filter. Add 4 mL of EM-grade 25 % glutaraldehyde and protect
from light. If the fixative is to be used the same day, keep it at
room temperature. If it will be used the next day, store at 4 °C
and filter before use.
2. Remove the medium from the culture plates, wash with PBS
and add the fixative (enough quantity to cover the cells).
However if many cells are detached, collect the medium and
centrifuge for 5 min to pellet the cells. Wash with PBS and
centrifuge again. Add the fixative and incubate 1-2 h at room
temperature, never less than 1 h. Centrifuge and remove the
fixative; then, add 1 mL of HEPES buffer. If the fixative was
added to the culture plate you will need to use a cell scraper to
detach and collect the cells. Centrifuge, remove the fixative,
and add 1 mL of HEPES to the cell pellet. Store fixed cells at
4 °C until next step (
see
Note 18
).
3. Transfer the pellet to a 1.5 mL Eppendorf tube. Wash the cells
three times with 1.3 mL of HEPES for 15 min. Resuspend the
cells using a wooden toothpick. Centrifuge at low speed for
5 min (minimal speed to precipitate cells, approx. 2,352 ×
g
).
4.
En bloc
staining: Add 300 μL of 1 % OsO
4
in 0.8 % K
3
[Fe(CN)
6
]
to the pellet and resuspend with a wooden toothpick. Incubate
for 1 h at 4 °C, protected from the light. Wash three times with
HEPES and centrifuge at low speed for 5 min. Add 300 μl of
2 % uranyl acetate and resuspend. Incubate for 40 min at 4 °C.
Wash three times with HEPES and centrifuge. To make sure
that all the staining agent is removed, incubate cells for 10 min
with HEPES between washes.
5. Dehydration: prepare dilutions of dry acetone at 50 %, 70 %,
and 90 % v/v in water. Add acetone 50 % to the cells, resus-
pend, and incubate for 15 min at 4 °C. Centrifuge at low speed
for 5 min. Remove the supernatant, add acetone 70 %, resus-
pend, incubate for 15 min at 4 °C, and centrifuge (process can
be paused for up to 1 h at this step if required). Repeat these
steps once with acetone 90 % and twice with acetone 100 %.
3.6.1 Fixation,
Dehydration, and
Embedding in Epoxy
Resins for Morphological
Studies
