Biology Reference
In-Depth Information
to a mica sheet and frozen ultrarapidly, followed by freeze-etching and generation of
cell surface replicas by oblique deposition of a heavy metal, usually platinum, and
strengthening with a layer of electron-translucent carbon. After removal of the bio-
logical material, the replica is mounted for examination in the transmission electron
microscope (TEM). The replica allows the analysis of the lateral distribution of re-
ceptors at the cell surface and offers planar views of large membrane areas, which
area not contacting the experimental support. Among the cell surface replication
methods, freeze-etching/freeze-drying is superior to critical point-drying or air-dried
membrane sheet generation in terms of structural fine detail preservation.
Unlike the superresolution light microscopy techniques, this technique permits
molecular resolution of cell surface molecules on the membranes that are not in con-
tact with the support, thereby avoiding visualization of potential clustering artifacts
induced by adherence (
James et al., 2011
). It can directly be applied to the cell sur-
face molecule of interest, without the need to genetically modify this protein with a
fluorophore, provided that good antibodies against the protein are available. The
technique can also be used to study ligand-induced receptor clustering or to analyze
the role of the cytoskeleton or particular lipid components of the membrane in recep-
tor clustering by pretreatment with the appropriate inhibitors or reagents (
Schamel
et al., 2005
). It is, however, not compatible with dynamic observations and, given the
relatively low labeling efficiency, does not allow absolute quantifications by direct
gold counting.
The protocol is based on our experience with primary lymphocytes and lymphocyte
cell lines that are labeled in solution before adherence to the mica sheet. This
freeze-etching replica technique was originally developed for adherent cell types,
and we refer the reader to this body of literature for a more extensive description of
this and related techniques (
Boonstra, van Bergen en Henegouwen, van Belzen,
Rijken, & Verkleij, 1991; Nermut, 1995; Severs, 1995; Severs, Newman, &
Shotton, 1995; van Belzen et al., 1988
).
21.1
MATERIALS
1.
Phosphate-buffered saline (PBS) pH 7.4
2.
PFA: 4% (w/v) solution in PBS (freshly prepared or from a frozen stock thawed
just before use)
3.
PBS containing 0.1% (w/v) bovine serum albumin (BSA), stored at 4
C
4.
100
g/ml poly-
L
-lysine in H
2
O
5.
Glutaraldehyde (25% solution EM grade, TAAB Laboratories, cat no. G011)
6.
Parafilm
7.
Bacterial culture dishes (10 cm diameter)
8.
24-well tissue culture plate
9.
Mica sheets (3
00
1
00
0.006
00
; e.g., TAAB, cat no. M054)
10.
Fine-tipped tweezers: Dumont tweezers (Dumoxel/Biology) curved or
straight tips
m
