Biology Reference
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13.4.3.2 Post-embedding method
This method has been used successfully to label phospholipids in samples that are
quick-frozen and freeze-substituted in uranyl acetate. Uranyl acetate is not likely
to react with lipid esters, but the LD core remains in the section at least partially
( Fig. 13.5 A) ( Ariotti et al., 2012 ; R. Parton, personal communication). This may
be because embedding material infiltrates and polymerizes at low temperature, so
that the structure is stabilized physically.
13.4.3.3 Ultrathin cryosectioning method
13.4.3.3.1 Tokuyasu method
For many purposes, this is the method of choice because of the following advantages:
(1) antibody labeling generally occurs with a high efficiency because the antigen is
not embedded in resin; (2) the bilayer membrane structure is observed clearly; (3) the
whole procedure (i.e., sample fixation to EM observation) can be completed quickly
(e.g., within a day).
On the other hand, because cellular structures are directly exposed to various so-
lutions without embedding resin, those that are not stabilized by chemical fixation
may be lost or dislocated. This is an actual problem for LDs, because the lipid ester
core is not fixed by aldehyde. Thus, in cryosections, the LD is often lost leaving an
artificial vacancy behind. Nonetheless, labeling for LD-associated proteins is usually
found at the rim of the vacant area, probably because some, if not all, proteins remain
by being cross-linked to the surrounding cytoplasmic matrix ( Fig. 13.5 B). In some
cases, the dislodged LD, seen as a round disk in ultrathin sections, adheres to a neigh-
boring area and labeling of LD-associated proteins can also be observed there. This is
an artifact and caution is necessary not to interpret it as indicating the true distribu-
tion of those proteins.
13.4.3.3.2 Methods using uranyl acetate
Use of uranyl acetate in the pick-up solution has been shown to increase retention of
membrane cholesterol in thawed cryosections ( Mobius et al., 2002 ), but it is not
likely to increase the chance of LD core preservation. However, by adhering cryo-
sections to EM grids electrostatically before thawing, LDs have been found to be
preserved better than in conventional Tokuyasu cryosections (R. Mesman, personal
communication). Lipid esters probably remain on the supporting membrane at least
partially, even after thawing.
Osmium tetroxide is generally precluded as a fixative for immunoEM, but it may
be used in the cryosection labeling methods. In the RHM method ( van Donselaar
et al., 2007 ), at least some proteins could be immunolabeled if osmium tetroxide
is rinsed off before the temperature goes above -50 C (E. van Donselaar, personal
communication). This is probably because the oxidizing effect of osmium tetroxide
is low and less destructive to antigenicity at low temperature. In the VIS2FIX H
method, by use of osmium tetroxide, the LD core is preserved ( Fig. 13.5 C) and sur-
prisingly some antigens can be labeled even after the osmium tetroxide treatment
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