Biology Reference
In-Depth Information
These techniques are further combined with electron tomography
(ET) to visualize subcellular structures, including microtubule
organization [
8
-
10
], endomembrane compartments [
11
-
13
], and
coated vesicles [
14
,
15
], and to analyze these structures in three
dimensions at nanometer-level resolutions.
Despite the advantages of HPF and FS, the protocols for indi-
vidual samples or for particular organelles need to be optimized
because there is no single HPF or FS protocol that works for every
tissue sample. For example, ultrastructural visualization of micro-
fi laments has been diffi cult, although they have been reported in
some cell types [
16
]. Although an improved freeze substitution
method has been developed, using tannic acid after treatment with
a mixture of OsO
4
and uranyl acetate (UA) during the FS process
[
17
], this method appears to be successful only with limited cell or
tissue types. Murata et al. optimized the conditions for visualizing
microfi laments by treating with 2 % OsO
4
, followed by 5 % UA at
−40 °C [
18
]. The improved visualization of microfi laments made
it possible to examine microtubules and coated pits in combination
with microfi laments in the cortical cytoplasm, facilitating accurate
ultrastructural analyses of microtubule ends [
18
], clathrin-coated
pits, clathrin-coated vesicles [
15
], and multivesicular bodies [
19
].
Processing samples for TEM by HPF and FS involves a num-
ber of steps which require more than several days. There are many
small details that researchers have to pay attention to in order to
achieve successful results. However, the amount and quality of
structural information acquired from well-frozen cell samples is
worth the effort and time. For example, budding of COPII vesicles
inside a ribosome-excluding scaffold [
12
], formation of clathrin-
coated vesicles from the plasma membrane under the preprophase
band [
15
], and the distinctive morphology of the plus-end of
phragmoplast microtubules were all observed in plant cell samples
processed by the combined use of HPF and FS [
8
]. Cryofi xation is
gaining popularity for TEM analysis, not only of Arabidopsis but
also of other plant species as more researchers realize its superiority
over chemical fi xation in capturing detailed and transient cellular
structures [
20
-
23
]. Procedures for Arabidopsis samples are avail-
able in other reports [
10
,
13
,
24
]. Here we describe procedures of
HPF and FS for non-Arabidopsis materials, including onion seed-
lings, BY-2 suspension-cultured cells, and maize endosperm cells.
2
Materials
2.1 High-Pressure
Freezing
1. High-pressure freezer, BAL-TEC HPM 010 (Boeckeler,
Tucson, AZ) or Leica HPM100 (Leica Microsystems, Buffalo
Grove, IL).
2. Specimen carrier: brass type A and type B planchettes (Ted
Pella, Redding, CA) or aluminum type A and type B planch-
ettes (Technotrade International, Manchester, NH).
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