Biomedical Engineering Reference
In-Depth Information
the cell of interest. Finally, the pattern of cell culture could be
used for the labeling of the cell position.
8. Observe the dynamics of the GFP-labeled structures in the
selected living cell using a multiphoton—a laser scanning con-
focal—or a digitalized fl uorescent-inverted microscope, which
allows the grabbing of a time-lapse series of images by a
computer.
9. At the moment of interest, add fi xative A to the cell culture
medium while still grabbing images (fi xative A: medium vol-
ume ratio is 1:1). Fixation usually induces the fast fading of
GFP fl uorescence and blocks the motion of labeled structures
in the cell.
10. Stop grabbing time-lapse images and keep the cells in fi xative
for 5-10 min (during this time, it is useful to grab a
Z
-series of
images of the cell).
11. Replace the mixture with the fi xative 1 and keep the cells in the
fi xative 1 for 5 min.
12. Replace fi xative 1 with fi xative 2 and keep cells there for
30 min.
13. Wash with 0.2 M HEPES (pH 7.2-7.3) for 10 min (
see
Note 1
).
1. Fix cells (
see
above).
2. Wash.
3. Incubate cells in the block solution.
4. Incubate with the fi rst primary antibody.
5. Wash.
6. Incubate with the secondary antibody against the fi rst primary
one.
7. Wash.
8. Incubate with the second primary antibody.
9. Wash.
10. Incubate with the second secondary antibody against the
second primary antibody and so on.
11. Photo cells after labeling with four different antibodies and
prepare them for EM.
3.2 Correlation
of Video Live Imaging
and Additional
Immune Labeling
of the Same Cell
3.3 Immune
Labeling for EM
with NANOGOLD
1. Wash the fi xed cells for 3 × 5 min with 0.2 M HEPES
(pH 7.2-7.3).
2. Incubate the cells with the blocking solution for 30 min and
then with the primary antibodies diluted in blocking solution,
overnight.
3. Wash the cells for 6 × 2 min with 0.2 M HEPES (PH
7.2-7.3).
