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13.4.3 Results and considerations
13.4.3.1 Pre-embedding method
This is the easiest way of doing immunoEM for people who have little experience in
EM. Up to the secondary antibody labeling step, it can be done in the same way as
that of immunofluorescence microscopy, and the latter procedure is the same as con-
ventional TEM sample preparation except for silver (gold) enhancement. The quality
of ultrastructure is directly correlated with the strength of fixation. Use of even a low
concentration of glutaraldehyde (e.g., 0.05%) helps preserve the structure of LDs and
other organelles significantly ( Fig. 13.4 ).
Saponin and digitonin do not permeabilize sterol-poor membranes such as the
endoplasmic reticulum membrane. Thus, antigens in the lumen of the endoplasmic
reticulum are not labeled except in the specialized subcompartment where sterol is
present in the limiting membrane ( Ohsaki, Cheng, Suzuki, Fujita, & Fujimoto, 2008 ).
FIGURE 13.4
Pre-embedding immunoEM. (A) Huh7 cells treated with DHA are fixed with 3% formaldehyde
in 0.1 M phosphate buffer for 15 min and permeabilized with 0.01% digitonin in PBS for
30 min. They are labeled simultaneously with anti-apolipoprotein B (ApoB) antibody (green),
anti-calreticulin antibody (red), and BODIPY593/603 (blue). Note that ApoB and calreticulin
are labeled in a crescent form on the LD surface (arrows). (B) Cells treated similarly as in
(A) are labeled with anti-ApoB antibody followed by Nanogold-conjugated secondary
antibody. The sample is subject to gold enhancement to make labels be observable by
TEM. The labels are observed within the lumen of thin membrane cisterns fused to
LDs (arrows).
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