Biology Reference
In-Depth Information
4.
Fix the samples with 2.5% glutaraldehyde in 1
PBS for 10 min at room
temperature and wash with distilled water.
5.
Enhance gold staining signal with a silver enhancement kit according to
manufacturer's instructions (Aurion) and wash with distilled water.
6.
Postfix the samples in 1% OsO4 containing 1.5% potassium ferrocyanide in 1
PBS for 30 min at room temperature and wash with distilled water.
7.
Dehydrate the samples in a series of ethanol washes (30%, 50%, 70%, 80%,
90%, 95%, and 100%, room temperature, 15 min each).
8.
Embed the samples in epoxy resin at room temperature for 2 days, followed by
polymerization at 60
C for 2 days.
9.
Cut 50-70-nm thick sections, transfer to formvar-coated grids and stained with
aqueous uranyl acetate (1%) and lead citrate (1%) for 6-10 min at room
temperature.
10.
Analyze sections on a FEI Tecnai 12 BioTwin transmission electron microscope.
SUMMARY
The robust method for generating single copy transgenes (
Frokjaer-Jensen et al.,
2012
), and the identification of hundreds of lipid droplet associated proteins by pro-
teomics (
Zhang et al., 2012
), provides exciting opportunities for many new lipid
droplet markers in
C. elegans
. New technologies have been developed for long-term
culturing and time-lapse imaging (
Krajniak & Lu, 2010
). It should be possible to ad-
dress questions on how the number and size of lipid droplets are regulated in unprec-
edented detail, all in the context of a live animal. Correlative fluorescence electron
microscopy offers exciting opportunities to pinpoint protein localization at the nano-
meter scale (
Watanabe et al., 2011
). Given the close proximity of lipid droplets with
other organelles such as the endoplasmic reticulum (
Blanchette-Mackie et al., 1995;
Jacquier et al., 2011; Robenek et al., 2009; Wolinski, Kolb, Hermann, Koning, &
Kohlwein, 2011; Xu et al., 2012
), nanoscopy should definitively address the local-
ization of proteins at the interface of two organelles.
Acknowledgments
The author thanks Sean McKinney for assistance with
Fig. 3.2
, Winfried Wiegraebe and
Fengli Guo for critical reading of the manuscript. This work was supported by the Stowers
Institute for Medical Research.
References
