Lipid Droplets and Viral Infections - Methods in Cell Biology - page 171

5. A total of 22 fractions (500 m l each) are collected from top to bottom.
6. The fractions are mixed 1:1 with Laemmli buffer for SDS-PAGE.
Quantification of protein expression in western blots is done using ImageJ
software.
10.2.2 Immunomicroscopy
10.2.2.1 Fixation and labeling
For fixation of cells, PFA is preferable over methanol as the latter disrupts LDs. Pro-
tocols for fixation with PFA differ, as proteins at the LD interface can be sensitive to
the concentration of PFA used. Therefore, the percentage of PFA should be opti-
mized for each experiment.
A representative example for each of the LD-staining methods commonly used in
the laboratory is shown in HCV-infected hepatoma cells costained for HCV core pro-
tein ( Fig. 10.4 ). Details about the protocols and outcomes for each labeling method
follow.
10.2.2.2 HCS LipidTOX neutral lipid stain
1. For staining of LDs using LipidTOX Red or Far Red, the reagent is diluted
1/200-1/1000 in PBS.
2. Permeabilized cells are incubated for 30 min with the diluted solution and
washed four times with PBS.
The concentration of LipidTOX can be adapted to the cell type and baseline levels of
LDs to minimize unwanted signal; when few LDs are present, the background fluo-
rescence with LipidTOX Red increases significantly.
10.2.2.3 Bodipy
1. For staining of LDs using Bodipy 493/503, the reagent is diluted 1/200 in PBS
(final concentration of 1 m g/ml).
2. Permeabilized cells are incubated for 10 min with the diluted solution and
washed three times with PBS.
The major disadvantage of Bodipy signal is that it bleaches fast. This can be prob-
lematic when quantifying LDs by immunomicroscopy as some signals (small LDs)
may be missed.
10.2.2.4 Oil-Red-O
1. For ORO staining, cover slips are incubated for 5 min in 60% isopropanol.
2. Cover slips are stained 10-20 min with ORO staining solution (the stock (at 0.5
% in isopropanol) is diluted 6:4 (stock:water) and filtered before use),
differentiated in 60% isopropanol for 1 s, and washed once with water.
ORO is probably the strongest and most complete stain for LD, but its emitted fluo-
rescence can bleed through other channels.