Biology Reference
In-Depth Information
10.
Gelatin capsules (Size 0, Agar Scientific AGG29210)
11.
Copper mesh sample grids, coated with formvar (Agar Scientific R1202). We
routinely use 200 hexagonal mesh grids (TAABGG017/C), but 100 mesh or slot
grids can also be used. Beware, as the larger the gaps between the copper bars,
the more easily the sample will distort and can tear
12.
High precision tweezers. We prefer self-closing tweezers as they facilitate the
handling of sample grids
13.
5% (w/v) uranyl acetate (Agar Scientific R1260A) in 50% ethanol
14.
Reynold's lead citrate solution (see
Reynolds 1963
)
20.2
METHODS
20.2.1
Cell transfection and observation
Cells are seeded into CLEM dishes that contain a coordinate-engraved glass cover-
slip, providing a pattern to be left in the base of the resin, once embedded. The
coordinates are essential for the LM to EM transfer as they allow cells of interest
to be tracked throughout the entire CLEM process.
Seeding the appropriate amount of cells into the dishes is important: too many
will make locating the cell of interest among many other unwanted cells difficult
once the sample is embedded in resin; it will also make reading the coordinates under
the light microscope difficult. However, seeding too few cells reduces the chances of
finding a suitable cell of interest. We, therefore, seed cells at 5% density, or 40,000
HeLa cells per 35-mm dish in preparation for imaging and resin-embedding the sam-
ple the following day. If the cells require transfection for over 24 h, we usually trans-
fect in separate plates (such as 6-well plates) and reseed them into the CLEM dishes
at the appropriate time to attain the required density. Aim for a cell density of
10-15% on the day of processing for CLEM.
20.2.2
Fixation and sample preparation
20.2.2.1
Fixative solution osmolarity
The physiological osmolality of mammalian tissue is
290 mOsm, depending on spe-
cies, tissue type, and hydration status (
Loqman, Bush, Farquharson, & Hall, 2010;
Mathieu, Claassen, & Weibel, 1978
). Fixative solutions should mimic physiological
osmolality, providing an iso-osmotic equilibrium between intracellular and extracel-
lular fluids.
Figure 20.2
shows the examples of orthogonally sectioned cells fixed with
solutions of varying osmotic strengths. At 440 and 1100 mOsm, a large amount of cell
shrinkage can be observed, with poor spindle apparatus preservation and unusually
dense cytosol. Therefore, we routinely use a fixing solution of
280 mOsm, consisting
of 3% glutaraldehyde, 0.5% paraformaldehyde in 0.05 M PB.
