Biology Reference
In-Depth Information
2.
Transfer the samples under liquid nitrogen to a Leica AFS unit, perform freeze-
substitution in 2% osmium tetroxide/0.1% uranyl acetate/acetone.
3.
Keep the samples for 24 h at
85
C and then hold at the following temperatures:
60
C (17 h); 0
C (2 h). The rate of temperature increase is set at 5
C/h.
4.
Bring the samples to room temperature in 1 h, followed by three changes of
absolute acetone.
5.
Infiltrate the samples with increasing concentrations of Epon over several days.
6.
Polymerize the samples at 60
C for 2 days.
7.
Section the samples at 70 nm and place them on formvar-coated slot grids.
8.
Stain sections with uranyl acetate (1%) and lead citrate (1%) for 6-10 min at
room temperature.
9.
Image stained sections on an FEI Tecnai 12 BioTwin transmission electron
microscope.
3.5
VISUALIZATION OF LIPID DROPLET ASSOCIATED
PROTEINS BY IMMUNOELECTRON MICROSCOPY
3.5.1
Background
A number of methods were attempted to visualize lipid droplet associated proteins by
immunoelectron microscopy (immuno-EM) in
C. elegans
samples. The standard
post-embedding labeling gave poor signals because the resin for embedding limited
epitope accessibility. Ultra-cryo on-section immunogold labeling allowed better epi-
tope presentation, but the lipid droplet morphology was poorly preserved. To over-
come these deficiencies, we adopted a method that was initially devised for
immunostaining of dissected intestine (
Francis, Barton, Kimble, & Schedl, 1995
).
Pre-embed immunogold labeling of dissected intestine combined with silver en-
hancement preserved the morphology of lipid droplets and gave strong specific sig-
nals. We were able to detect GFP::DGAT-2 in the vicinity of the lipid droplet surface
(
Xu et al., 2012
).
3.5.2
Methods
We performed immuno-EM on transgenic animals that carry the single copy trans-
gene for GFP::DGAT-2 expression.
1.
Fix dissected
C. elegans
intestines with 2% formaldehyde for 4 h at room
temperature.
2.
Permeabilize the samples by incubation with 0.1% Triton X-100 in 1
PBS for
รพ
1% BSA for 1 h at room temperature.
3.
Incubate the samples with primary and secondary antibodies at 4
C for 16 h
with gentle rocking. Use anti-GFP antibody (Invitrogen, mAb 3E6, 1:200) and
goat-anti-mouse super small gold IgG antibody (Electron Microscopy Sciences,
25121, 1:50) as primary and secondary antibodies, respectively.
15 min and block in 1
PBS
