Biology Reference
In-Depth Information
8000
g
for 5 min before use to remove unwanted precipitate. Grids should be dried
face-up for at least 2 h on clean filter paper before imaging, and kept in a clean, dust-
free environment (such as a Petri dish or grid storage box).
20.2.4
Orthogonal sectioning
Orthogonal sectioning involves taking sections that are perpendicular to the spindle
axis. In mitotic cells, this is useful to view and quantify most K-fiber microtubules
within a single section. Quantifying K-fiber microtubules is possible using longitu-
dinal serial sections (
McEwen, Heagle, Cassels, Buttle, & Rieder, 1997
); however,
we avoided this method because (1) serial sections are particularly difficult to ac-
quire and (2) spatial distribution analysis cannot be performed, as the compression
forces exerted on each section of a serial reconstruction by the knife will likely de-
form the sample more than a single orthogonal cross-section through a K-fiber.
Figure 20.4
contains a pictorial guide of the steps required to prepare the sample
for orthogonal sectioning. The samples to be sectioned orthogonally should consist
of a flat layer of resin (without the resin capsule;
Fig. 20.4
A). Once removed from the
oven, separate the dish from the resin using the same method as for the longitudinal
samples (see above) and with the same amount of care (
Fig. 20.4
B-D). Using a tissue
dissection microscope, find the coordinate containing your cell of interest and circle
it using a marker pen (
Fig. 20.4
H). Using the coordinate grid and the LM images
taken previously as references (
Fig. 20.4
E-G), determine the position and direction
of the spindle axis and draw an elongated rectangle around the cell of interest
(
Fig. 20.4
H-J). Cut out the rectangle using the hacksaw (
Fig. 20.4
K and L), paying
attention not to touch or damage the surface of the marked coordinate. You should
end up with a strip of resin (
Fig. 20.4
M). Carefully remove excess off one end, so the
cell is near the tip, and insert it into a microtome chuck (
Fig. 20.4
N and O).
Using an ultramicrotome, you should be able to see the coordinates and can trim
excess resin from the tip using glass knives until you approach the cell of interest
(
Fig. 20.4
P-R). Trim away resin from either side of the cell
to a depth of
m buffer zone around the cell. Finally, trim away
excess resin from the “upper” side of the strip, which is the block face positioned
reverse-parallel to the one imprinted with coordinates. The thickness should be sim-
ilar to the width on either side of the cell edge so that the block face is square shaped.
The cell can now be sectioned using a diamond knife; we routinely take 80-100 nm
slices. Sections should be collected and treated as described for longitudinal section-
ing (see above), along with the same poststaining method.
100
m
m, leaving a 50- to 100-
m
20.2.5
Imaging and sample tilting
In longitudinal sections, K-fibers can be identified as bundles of microtubules in par-
allel conformation terminating at the kinetochore. During image capture, we typi-
cally take 4
m
mby3
m
m images at 60,000
magnification. This allows us to
distinguish adjacent microtubules and the material that cross-links them with enough
