Biology Reference
In-Depth Information
other fixatives, such as uranyl acetate, tannic acid, and glutaraldehyde, are used in
various combinations. Ultrathin sections are counterstained with uranyl acetate and
lead nitrate.
13.3.3
Results and considerations
Freeze-substitution in combination with quick-freezing is widely used to prepare
samples for conventional TEM and EM tomography (
McIntosh, Nicastro, &
Mastronarde, 2005
). The method has a better chance of preserving lipid-rich struc-
tures than conventional methods because it can bypass the step of aldehyde fixation.
That is, when cells are fixed by aldehydes and processed by conventional methods,
artifacts like vesiculation and myelin figures occasionally form, probably because
aldehydes cross-link proteins while leaving most membrane lipids unfixed. This
may pose problems in studying LDs, because they are often associated very closely
with organelles such as the ER, mitochondrion, and peroxisome (
Fujimoto, Ohsaki,
Cheng, Suzuki, & Shinohara, 2008
).
It is not necessarily clear, however, how precisely freeze-substitution can immo-
bilize cellular macromolecules at the location where they are found in living cells and
visualize biological structures as they are in living cells. In fact, the image obtained
by freeze-substitution of quick-frozen samples is not identical with that obtained by
cryoelectron microscopy of frozen-hydrated sections (
Al-Amoudi et al., 2004
). This
may be partly because fixatives used in freeze-substitution begin to react with cel-
lular constituents only after the sample is warmed to a certain temperature, so that
some structures may go through some changes by the time they are stabilized
securely.
Another problem with freeze-substitution is that membranes do not show good
contrast in comparison with conventional methods. Several methods to improve
the membrane contrast have been developed, such as addition of a small amount
of water to the fixative (
Walther & Ziegler, 2002
) and use of tannic acid-mediated
osmium impregnation (
Jimenez et al., 2009
). Nonetheless, it is sometimes difficult to
observe the unit membrane structure unambiguously. Tomographic observation
of quick-frozen, freeze-substituted specimens may provide more information by
removing this obstacle.
13.4
IMMUNOELECTRON MICROSCOPY OF LD-ASSOCIATED
PROTEINS
13.4.1
Rationale
Immunoelectron microscopic methods are often classified based on whether anti-
bodies are applied to cells (and tissues) before or after ultrathin sectioning. The for-
mer method is often referred to as the “pre-embedding labeling method,” because
cells are treated with antibodies before resin embedding. The latter category includes
