Biology Reference
In-Depth Information
(
Liou, Geuze, & Slot, 1996
). In an alternative method, cells are quick-frozen and
freeze-substituted in a solution containing uranyl acetate; they are then rehydrated
and cryosections are prepared as in the Tokuyasu method (
van Donselaar,
Posthuma, Zeuschner, Humbel, & Slot, 2007
). In a third method, cells are quick-frozen
and cryosectioned without any pretreatment such as fixation or sucrose infiltration;
the obtained ultrathin cryosections are put onto a frozen uranyl acetate solution
before thawing (
Karreman, van Donselaar, Gerritsen, Verrips, & Verkleij, 2011
).
Lipid esters can be stabilized by osmium tetroxide. Although the osmium solu-
tion is generally incompatible with immunocytochemistry due to a strong oxidizing
effect, it may be used for some antigens in combination with a cryosectioning method
(
van Donselaar et al., 2007
).
13.4.2
Methods
13.4.2.1
Choice of fixation protocol
In fixing cells for immunoEM, appropriate fixation needs to be determined for each
antigen by testing several different protocols. Formaldehyde is a relatively weak fix-
ative, but it can quickly penetrate into the cell and does not harm the antigenicity
significantly. Glutaraldehyde is much better than formaldehyde in preserving the ul-
trastructure, but it is slow in penetration and decreases the labeling intensity of most
antigens.
The optimum protocol needs to be selected by looking at results of immunoEM,
but immunofluorescence labeling can be utilized to give a starting point. As a first
step, for example, cells are fixed with 3% formaldehyde in a neutral buffer (e.g.,
0.1 M sodium phosphate buffer, pH 7.4) for 30 min and labeled for immunofluores-
cence microscopy. If intense labeling is observed by this fixation, an increasing con-
centration of glutaraldehyde should be added to see whether sufficient labeling
persists. (Note: samples after fixation may need to be treated with 1 mg/ml sodium
borohydride in 0.1 M Tris-buffered saline (pH 8.2) for 5 min to quench autofluores-
cence of glutaraldehyde.) The highest concentration of glutaraldehyde that does not
decrease the labeling significantly may be chosen as a protocol for immunoEM.
Some LD-associated proteins show unique behavior to fixation (
Ohsaki, Maeda,
& Fujimoto, 2005
). That is, they are not labeled when cells are fixed with formal-
dehyde alone, whereas intense labeling occurs when glutaraldehyde is added to
the fixative. This is probably because formaldehyde alone does not provide sufficient
cross-linking to immobilize the antigens to the LD surface or to the surrounding cy-
toplasmic matrix and they are dissolved by the subsequent Triton X-100 treatment.
13.4.2.2
Pre-embedding method
Cells after appropriate aldehyde fixation are treated with 0.2% saponin (or 0.01% dig-
itonin) in PBS for 30 min at room temperature. After residual aldehydes are quenched
by 10 mMglycine in PBS, cells are blockedwith 3%BSAor other appropriate blocking
solution in PBS for more than 10 min, and incubated overnight at 4
Cusingaprimary
antibody in PBSwith 1%BSA containing 0.1% saponin. After repeated rinses, they are
