Biology Reference
In-Depth Information
methods using resin-embedded sections, which is referred to as the post-embedding
labeling method, and a method using ultrathin cryosections.
Despite the aforementioned insufficiency of aldehyde fixation, a combination of
formaldehyde and glutaraldehyde is often used to fix samples for immunoelectron
microscopy. Quick-freezing methods can be used to avoid the problem of aldehyde
fixation, but sophisticated methods and equipment are required for processing
samples.
13.4.1.1
Pre-embedding method
In order to label intracellular molecules with antibodies without sectioning, the
plasma membrane needs to be permeabilized. For this purpose, cells are often treated
with a low concentration of saponin or digitonin after aldehyde fixation. Saponin and
digitonin primarily work on sterols so that the membrane structure can largely be
preserved (
Elias, Goerke, Friend, & Brown, 1978
). In this respect, they are superior
to detergents like Triton X-100 and Nonidet P40, which destroy the membrane struc-
ture almost completely by extracting lipids indiscriminately.
For this method, antibodies conjugated to small golds (i.e., small gold atom clus-
ters
or ultra-small colloidal gold) are generally used because they can easily pene-
trate the pores made by saponin and digitonin. After labeling, metallic silver (or gold)
particles are developed around the small golds to make them clearly observable by
conventional TEM.
Colloidal gold particles of 5-15 nm in diameter, which are used as markers for
other immunoEMmethods, are not appropriate for the pre-embedding method due to
their large size.
13.4.1.2
Post-embedding method
When cells are fixed with aldehydes alone and dehydrated with organic solvents for
resin embedding, lipids and membrane structures are not preserved. This problem is
circumvented by quick-freezing samples without aldehyde fixation and freeze-
substituting them in uranyl acetate. Infiltration and polymerization of embedding
resin at a very low temperature further helps preserve the structure and antigenicity.
13.4.1.3
Ultrathin cryosectioning method
In the original Tokuyasu method, cells fixed with aldehydes are infiltrated with a
high concentration of sucrose, frozen with liquid nitrogen, and ultrathin cryosections
are prepared (
Slot & Geuze, 2007; Tokuyasu, 1973
). Because the samples are not
dehydrated, the LD structure should be maintained up to the sectioning step. But
when sections are thawed and subject to immunolabeling procedures, unfixed lipids
are directly exposed to aqueous buffers and are likely to be lost or dislocated.
Use of uranyl acetate at a certain step helps preserve membrane structure by
binding to the phosphate group of phospholipids (
Ginsburg & Wolosin, 1979;
Huang, Blume, Das Gupta, & Griffin, 1988
). Three different methods have been
reported. A method closest to the original Tokuyasu method uses cells fixed with
aldehydes and retrieves cryosections with a solution containing uranyl acetate
