Biology Reference
In-Depth Information
and mark the approximate location of the cell with a fine marker pen on the underside
of the dish.
20.2.2.3
Resin embedding
Cells become round during mitosis and are, therefore, less adherent to their substrate.
This means that during all steps up to resin embedding, dishes must be handled with
extreme care so as to not detach or change the orientation of the cell.
Next, replace the wash solution with a few drops of 1% osmium tetroxide on the
coverslip for 1 h. Remove the osmium and gently rinse the cells twice for 30 min
with double-distilled water. Remove the water and replace with 30% ethanol for
30 min, then replace with a small amount of 0.5% uranyl acetate in 30% ethanol
for 1 h. Next, the cells need to be dehydrated using a gradient of sequential solutions
containing increasing amounts of ethanol. Replace stepwise with each of the follow-
ing solutions: 30%, 50%, 60%, 70%, 80%, and 90% ethanol, then twice with 100%
ethanol, incubating at each step for 10 min.
The cells can now be infiltrated with resin. Mix a 1:2 ratio of resin:ethanol so-
lution, making sure that it is fully homogenized using a 3-ml plastic Pasteur pipette.
Remove the ethanol from the dish and lightly cover the bottom of the dish with the
resin infiltration mix for 20 min. Remove and replace with a 1:1 ratio of resin:ethanol
solution for 20 min. Remove the mix and replace with a
2-mm layer of 100% resin
covering the bottom of the dish. If the sample is to be sectioned orthogonally, the dish
can be placed in a 60
C oven for 48-72 h. For longitudinal sectioning, fill either half
of an embedding capsule with 100% resin and gently place it open-side down onto
the cell (
Fig. 20.3
A), which you should be able to locate using the pen mark placed
earlier. The dish can then be placed in the oven.
We recommend the use of EPON resin as other resin types (e.g., Agar Scientific
Low Viscosity Resin) that we have tested react and bind the CLEM dish, making the
separation steps (below) much more difficult.
20.2.3
Longitudinal sectioning
Longitudinal sectioning is the conventional EM method for viewing cells. Sections
parallel to the plane of the coverslip are taken from the base of the cell moving pro-
gressively upward (see
Fig. 20.1
C). This plane of sectioning allows extended lengths
of microtubules to be observed and is, therefore, particularly useful for analyzing
microtubule attachment to the kinetochore, or quantifying microtubule cross-linkers
(
Fig. 20.1
D).
Once the resin has fully polymerized, the dishes can be removed from the oven.
Figure 20.3
contains a pictorial guide of the steps required to separate the resin and
dish up to the sectioning. Using pliers, start by cutting-off the edges of the dish en-
tirely (
Fig. 20.3
A-C) so that the seam between resin and plastic is accessible all the
way around the dish. Very carefully insert a razor blade between the plastic and resin
(
Fig. 20.3
D), slowly forcing the razor toward the center of the dish all the way around
