Biology Reference
In-Depth Information
30 min, so pinpointing the exact stage of the cell cycle for a particular cell is
critical before engaging in time-costly EM sample preparation. This is why the
ability to observe and select cells of interest using LM prior to EM sample
processing is a great advantage; allowing both the stages of mitosis to be chosen
carefully, and to ensure that the cell is adequately expressing a fluorescent protein
of interest.
Studies using EM to research mitotic spindles have yielded outstanding data,
such as the quantification of microtubule polarity by
Euteneuer and McIntosh
(1981)
, the study of microtubule spacing, position, displacement, and length
(
McDonald, O'Toole, Mastronarde, & McIntosh, 1992
), or the more recent
whole-cell reconstruction by electron tomography to study cytoskeletal elements
(
Hoog et al., 2007
).
Here, we describe our own application of CLEM to study the ultrastructure of
the mitotic spindle, particularly kinetochore fibers (K-fibers) (
Booth, Hood,
Prior, & Royle, 2011; Cheeseman, Booth, Hood, Prior, & Royle, 2011
). We
describe both longitudinal and orthogonal sectioning relative to the spindle axis
(
Fig. 20.1
), which reveal different information about spindle architecture
(
Fig. 20.1
D), and how we can quantify such results. Longitudinal sectioning
has allowed us to quantify microtubule cross-linkers between K-fiber
microtubules, whereas sample-tilting of orthogonally sectioned K-fibers allowed
the quantification of the number of microtubules forming the fiber. Subsequent anal-
ysis of the spacing of these microtubules allows us to measure their density and
distribution.
20.1
MATERIALS
1.
35-mm glass-bottomed dishes with etched coordinates (MatTek Corporation,
P35G-2-14-C-GRD)—referred to here as CLEM dishes
2.
0.1 M phosphate buffer (PB): mix 0.2 M Na
2
HPO
4
with 0.2 M NaH
2
PO
4
and
dilute to 0.1 M. Solution should be at pH 7.4
3.
Fix solution (EM grade fixatives: 3%, w/v, glutaraldehyde (Agar Scientific
R1020), 0.5%, w/v, paraformaldehyde (Agar Scientific R1026) in 0.05 M PB)
4.
Wash solution (0.05 M PB, 0.1 M sucrose)
5.
DNA stain solution (0.1%, w/v, Hoechst-33342 in wash solution, or other
similar DNA dye)
6.
1% osmium tetroxide (Agar Scientific R1015) in water
7.
0.5% (w/v) uranyl acetate (Agar Scientific R1260A) in 30% ethanol
8.
Molecular grade 100% ethanol (Sigma-Aldrich 270741-1L)
9.
EPON resin (Agar Scientific R1031, made up using the supplier's specifications
for a “medium” block. Make sure resin mix is fully homogenized, and
containing as few bubbles as possible. 200 ml of resin can be made up at a time,
aliquoted into small glass vials, and kept frozen at
20
C)
