Biology Reference
In-Depth Information
This step requires experience. First cut semithin sections (0.2-0.5
m), stain with to-
luidine blue O, observe the sections under light microscope, trim the block further if
needed. Then ultrathin sections (60-90 nm) are cut and collected with copper grids.
m
6.
Section staining and observation
Grids are first stained with 2% uranyl acetate for 3-5 min in dark at room temper-
ature, washed with distilled water to remove any unreacted uranyl acetate, and then
stained with 1% lead citrate for 20-30 min. Sections on grids are observed under a
JEM-1400 transmission electron microscope (JEOL Inc., Peabody, MA, USA).
5.3
ISOLATION AND PURIFICATION OF OIL DROPLETS
To further understand the structure of oil droplets and their associated proteins, oil
droplets need to be isolated and purified from the cells. The purified oil droplets are
supposed to maintain their integrity and display electrostatic repulsion and steric hin-
drance on their surface and the proteins nonspecifically associated with or trapped
within should be removed (
Tzen, Peng, Cheng, Chen, & Chiu, 1997
). However, ap-
parent contamination of subcellular preparations in
Chlamydomonas
seems to be a
common challenge, although in some cases it is not easy to distinguish the contam-
ination from the genuine association between oil droplets and other organelles
(
Moellering & Benning, 2010; Nguyen et al., 2011
). The protocol developed by
Nguyen et al. (2011)
appears to yield a highly purified oil body preparation as judged
by the oil content and the SDS-PAGE profile.
SUMMARY
Despite recent research efforts, oil droplets are understudied organelles and many
aspects of cell biology of oil droplets remain unknown, even in yeast and mammalian
model systems (
Farese & Walther, 2009; Walther & Farese, 2009, 2012
) and we are
just beginning to understand the dynamics and ontogeny of oil droplets in microalgae
(
Fan et al., 2011; Goodson et al., 2011
). The protocol described here provides a basic
guide for the imaging of oil droplets in living cells, lipid quantification, and genetic
mutant screens based on Nile red fluorescence staining and ultrastructural analysis of
oil droplet biogenesis, growth, and their interactions with other cellular compart-
ments by TEM.
Acknowledgments
This work was supported by the Division of Chemical Sciences, Geosciences, and Biosciences,
Office of Basic Energy Sciences of the U.S. Department of Energy. Use of the transmission elec-
tron microscope at the Center of Functional Nanomaterials was supported by the Office of Basic
Energy Sciences, U.S. Department of Energy, under Contract DEAC02-98CH10886.
