Imaging Lipid Droplets by Electron Microscopy - Methods in Cell Biology - page 239

( Karreman et al., 2011 ). The reason for this compatibility is not clear because os-
mium tetroxide is used at around 0 C.
Methods described in this section are intended to detect endogenous molecules,
but they can be also used to examine distribution of molecules tagged with geneti-
cally encoded molecules or those carrying artificial chemical groups ( Jao, Roth,
Welti, & Salic, 2009 ). To examine LD-related protein dynamics in a broad cellular
context, correlative light and EM using some genetically encoded tags may be
exploited ( Shu et al., 2011 ).
13.5 FREEZE-FRACTURE ELECTRON MICROSCOPY
13.5.1 Rationale
In freeze-fracture EM, frozen cell samples are fractured at a low temperature and a
thin layer of platinum and carbon is deposited onto the newly revealed surface by
vacuum evaporation to physically stabilize the underlying structure. The thin film
made of platinum and carbon, which is called freeze-fracture replica, is treated with
sodium hypochlorite to dissolve all biological materials for genuine morphological
observation. Because the fracture plane tends to run between the two membrane leaf-
lets, the method has become an indispensable tool in membrane biology ( Severs,
2007 ). If the replica is treated with sodium dodecyl sulfate (SDS) instead of sodium
hypochlorite, molecules cast by the platinum/carbon deposition are held stably in the
replica and can be labeled by appropriate probes for EM observation ( Fujimoto,
1995; Fujita, Cheng, & Fujimoto, 2010 ). This method has been used successfully
to label membrane proteins and lipids.
13.5.2 Method
Quick-frozen specimens are transferred to the cold stage of a freeze-fracture device
(BAF400 [BALTEC], JFD-II [JEOL], etc.). They are freeze-fractured at below -100º
and evaporated with platinum and carbon in a high vacuum. For genuine morpholog-
ical observation, platinum/carbon of 1-2 nm thickness is first evaporated at an angle
of 30-45º, followed by carbon of 20 nm thickness at a 90º angle. After thawing in
the atmosphere, the sample is treated with household bleach for more than 1 h to
digest biological materials, rinsed in distilled water, and picked up on grids for
EM observation.
For immunolabeling, vacuum evaporation in three consecutive layers (i.e.,
2-5 nm carbon, 1-2 nm platinum/carbon, and 10-20 nm carbon) is used, because
this gives better immunolabeling than the conventional two-layer method ( Fujita
et al., 2007; Hagiwara, Fukazawa, Deguchi-Tawarada, Ohtsuka, & Shigemoto,
2005 ). The thawed replica is treated with 2% SDS in PBS for 1 h to overnight at
60-70 C. They are rinsed with PBS containing 1% Triton X-100 (PBST), blocked
with 3% BSA in PBS, and incubated with a primary antibody in 1% BSA in PBST
at 4 C overnight. After rinsing, they are treated with colloidal gold-conjugated