Biology Reference
In-Depth Information
anti-immunoglobulin antibody or protein A in 1% BSA in PBST for 30 min at 37
C.
Finally, replicas are rinsed with distilled water and picked up on EM grids.
13.5.3
Results and considerations
Combined with quick-freezing, freeze-fracture enables observation of cellular struc-
tures with no intervention of chemicals. In the conventional freeze-fracture tech-
nique, specimens are usually treated with glycerol before freezing to suppress ice
crystal formation. But because glycerol may not be inert to phospholipids
(
Anchordoguy, Rudolph, Carpenter, & Crowe, 1987; Westh, 2003
), it is not war-
ranted that lipid-rich structures are not affected by the treatment.
When membranes are freeze-fractured, the fracture plane does not run along the
true surface, but it runs between the two leaflets of the phospholipid bilayer. This is
because the molecular interaction between the two phospholipid bilayers is at its
weakest in a frozen condition. With regards to LDs, however, it is not clear if the
fracture plane preferentially runs between the surface phospholipid monolayer
and the underlying lipid ester core. Occasionally, an LD is fractured in multiple
layers to show an “onion-skin” appearance, which may represent some layered struc-
ture in the lipid ester core. In many instances, LDs are cross-fractured and appear as
round structures in the freeze-fracture replica. Immunolabeling for LD-associated
proteins occurs in those structures specifically (
Fig. 13.6
). Labeling of some
FIGURE 13.6
ImmunoEM using freeze-fracture replicas. Freeze-fracture replicas of quick-frozen
HepG2 cells are treated with SDS and labeled by anti-PLIN2 antibody. The colloidal gold
labeling is observed along the periphery of a round structure, which represents a
cross-fractured LD. The Golgi membranes (G) are not labeled for PLIN2.
