Biology Reference
In-Depth Information
Wash again with PBSAT and leave a drop for 5 min while preparing the primary
antibody solution.
3.
Aspirate off the PBSAT and incubate with 100
l of primary antibody solution
for 1 h at room temperature (or overnight at 4
C). If using several antibodies,
they should be recognized by different secondary antibodies. For microtubules,
we use anti-
m
-tubulin B-5-1-2 antibodies.
4.
Wash with PBSAT and leave a drop while preparing the secondary antibody
solution. Aspirate off the PBSAT and add the secondary antibody for 30 min.
5.
Wash in PBSAT and then in PBS. Remove the PBS, add DAPI for 1 min, then
wash with ddH
2
O, and let air dry under a cover before mounting the coverslips on
slides with 20
a
m
l Mowiol.
More commonly used these days to label mitochondria are MitoTracker probes
(
www.invitrogen.com
)
, which are well adapted for imaging of fixed cells but should
not be incubated with the cells for longer than 15-30 min. At longer incubation
times, the MitoTracker starts to label other membrane structures and even disturbs
the native mitochondrial morphology, which makes it unsuitable to use in live-cell
imaging. The MitoTracker must be incubated with the cells prior to fixation.
7.4.2
Live-cell visualization
For live-cell imaging of mitochondria, we use Mito-protein, which is a gene-based
marker expressing a 29-amino acid-long polypeptide corresponding to the mitochon-
drial targeting sequence from human cytochrome
c
oxidase subunit 8. Mito-protein
can be tagged with mRFP or EGFP, giving strong signal and low bleaching. Fluo-
rescent Mito-protein can be used for transient transfection or to create stable cell
lines. Cells stably expressing fluorescent tubulin can also be made.
For live-cell imaging, the patterned coverslip needs to be mounted in a chamber
so that medium can be added onto the cells. Make sure that the size of the coverslips
and the chamber are compatible. Different kinds of chambers are commercially
available, for example, Ludin chamber (
www.lis.ch
) or magnetic chamber (
www.
chamlide.com
)
. We find the magnetic chamber to be very practical due to its quick
magnetic closing function and the fact that it exists for different sizes and also for
square coverslips, but the gasket might be a bit tricky to clean, which may stress
the cells if residual cleaning solvent remains. Letting the gasket incubate in the me-
dium overnight will help dilute any residual solvent.
It is possible to seed and spread cells on the coverslip directly inside the chamber
or to move the coverslip with the already spread cells from the well into the chamber.
In the latter case, make sure to have a pipette with medium ready before taking the
coverslip from the well to minimize the time in open air to prevent dehydration.
The patterned cells inside the chamber are now ready to be imaged live on the
microscope.
Figure 7.2
A compares a fixed cell spread on a nonpatterned coverslip
to that of a cell spread on a rectangular shape micropatterned coverslip.
Figure 7.2
B
shows the robustness and repeatability of the micropatterned cells.
