Biology Reference
In-Depth Information
incubated for 2-3 h at room temperature with an appropriate secondary antibody con-
jugatedwith a small gold atomcluster (Nanogold
TM
[Nanoprobes]) (
Hainfeld, 1987
)or
ultra-small colloidal gold (Aurion) (
van de Plas &Leunissen, 1993
) in the solution con-
taining saponin. Saponin (digitonin) is included in the antibody solution because mem-
brane pores may reseal during incubation. By using the secondary antibody doubly
conjugated with a small gold atom cluster and fluorochrome (Fluoronanogold
TM
[Nanoprobes]), the sample can be observed by fluorescence microscopy to check
whether sufficient labeling has occurred. The labeled samples are rinsed and fixed with
2.5% glutaraldehyde in PBS for more than 30 min to reinforce fixation.
After extensive washes with distilled water, samples are incubated with a silver or
gold development solution to increase the size of the gold label. This is a gold-
catalyzed reaction to reduce silver (or gold) ions to metallic silver (or gold), a process
similar to photographic development. The metal solutions can be prepared by mixing
ingredients (
Hayat, 1995
) or obtained commercially (HQ Silver
TM
, GoldEnhance
TM
[Nanoprobes], R-GENT SE-EM
TM
[Aurion]). The incubation time for development
needs to be adjusted so that the silver or gold particle can grow to an appropriate size;
for example, we allow 2-4 min for GoldEnhance.
The sample after the silver or gold development is treated with osmium tetroxide,
dehydrated, and embedded as with conventional TEM samples. The sample pro-
cessed with silver enhancement solution should not be incubated in the osmium te-
troxide solution for long, because the formed metallic silver may be lost due to strong
oxidization. Gold toning can be used to prevent the loss of the silver precipitate
(
Sawada & Esaki, 1994
).
13.4.2.3
Post-embedding method
Cells are subjected to high-pressure freezing and freeze-substituted in 0.2% uranyl
acetate in acetone containing 5% water and 4%methanol for 2 days at
85
C. After
50
C, they are washed and infiltrated with Lowicryl HM20 resin, and
polymerized by UV light for 2 days (
Fairn et al., 2011
). Ultrathin sections are cut by
conventional ultramicrotome, picked up onto EM grids, and labeled as with ultrathin
cryosections, except that the final on-grid staining step is omitted.
warming to
13.4.2.4
Ultrathin cryosectioning method
13.4.2.4.1
Tokuyasu method
Cells fixed with aldehydes are infiltrated with 2.3 M sucrose in PBS for 1 h to over-
night and frozen by plunging into liquid nitrogen. Ultrathin cryosections are cut by an
ultramicrotome with a cryo-attachment (e.g., Leica EM FC6). For most purposes, the
temperature is set around -110
C and sections of ca. 70 nm in thickness are pre-
pared. Ultrathin cryosections are picked up by using a drop of 1.15 M sucrose and
1% methylcellulose held on a wire loop, thawed, and transferred to a formvar mem-
brane on an EM grid.
Grids bearing ultrathin cryosections are rinsed with PBS containing 10 mM gly-
cine for 30 min and treated with 3% BSA or other appropriate blocking solution in
PBS for more than 10 min. They are incubated with a primary antibody solution for
