Biology Reference
In-Depth Information
30 min at 37
C (or overnight at 4
C, etc.), rinsed with 0.1% BSA in PBS, and trea-
ted in a secondary antibody solution in a similar manner. Depending on the kind of
primary antibodies, either protein A or anti-IgG antibody conjugated with colloidal
gold is used. Protein A-colloidal gold is preferred over anti-IgG-colloidal gold be-
cause of its stability and the 1:1 binding stoichiometry with IgG, but it does not bind
well with some immunoglobulin classes and subclasses. After rinses, grids are trea-
ted with buffered 2% glutaraldehyde for 10 min to stabilize the antigen-antibody
complex.
After rinses with distilled water, sections are adsorption-stained with neutral ura-
nyl acetate solution containing 2% uranyl acetate and 0.15 M oxalic acid for 10 min,
rinsed again, and incubated with a mixture of 2%methylcellulose and 0.1-0.5% ura-
nyl acetate, and dried on a wire loop.
13.4.2.4.2
Methods using uranyl acetate
i.
Inclusion of uranyl acetate in the pick-up solution (
Liou et al., 1996
)
Cryosections are prepared as the original Tokuyasu method except that phosphate
buffer is not used. They are picked up by a mixture of 2% methylcellulose and
0.3-3% uranyl acetate and rinsed with distilled water. The subsequent solutions
for immunolabeling procedures need to be prepared in 0.1 M PIPES buffer
(pH 7.4) instead of PBS.
ii.
Freeze-substitution and rehydration before cryosectioning (the RHM method)
(
van Donselaar et al., 2007
)
Cells are quick-frozen and freeze-substituted in anhydrous acetone. Either 0.2% ura-
nyl acetate, 0.5% glutaraldehyde, or 1-2% osmium tetroxide can be added to the so-
lution. After the rinses, they are warmed, rinsed, and rehydrated at
30
Cby
incubating sequentially in 95% acetone in water, 90% acetone in water, 80% acetone
in water, 70% acetone in water, 50% acetone in PHEM buffer (60 mM PIPES,
25 mM HEPES, 10 mM EGTA, and 2 mM MgCl
2
, pH 6.9), and 30% acetone in
PHEM buffer for 10 min each. They are washed in PHEM buffer and infiltrated with
2.3 M sucrose in PHEM buffer before freezing and sectioning as in the original
Tokuyasu method.
iii.
Quick-freezing and rehydration after cryosectioning (the VIS2FIX
H
method)
(
Karreman et al., 2011
)
Ultrathin cryosections are cut directly from quick-frozen samples without any treat-
ment and adhered to EM grids electrostatically as in cryoelectron microscopy of vit-
reous sections (
Al-Amoudi et al., 2004
). They are placed at
90
C on frozen
fixative consisting of 0-0.5% osmium tetroxide, 0.2% uranyl acetate, and 0-0.2%
glutaraldehyde in PHEM buffer. After melting on a hotplate, the sections are kept
on the fixative solution for 10 min on ice. They are rinsed with PHEM buffer before
immunolabeling.
