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and subcellular fractionation procedures, then this provides strong evidence that an-
tibody labeling is specific. In this way other techniques are a valuable complement to
cell imaging.
12.3.2
Placing fat droplets in intracellular context
In addition to authentic, well-defined signals, useful images of fat droplets require
subcellular context. For example, perilipin 3 staining in fat storing OP9 cells reveals
only puncta (
Fig. 12.1
) and round hollow appearing structures (
Fig. 12.3
). This stain-
ing pattern by itself is uninformative, because the cell boundaries are indiscernible,
as is the intracellular distribution. However, imaging the ER reveals that the nuclear
envelope and the ER extend to the cell periphery, and delineates the cell boundaries.
In this context, we see that the perilipin 3 puncta at the periphery fall along the
smooth ER (
Fig. 12.1
). We also see that the majority of the perilipin 3 puncta are
in the perinuclear region. Several factors contribute to interpreting these observa-
tions. First, the signal from this anti-perilipin 3 antibody is unambiguous, because
its authenticity has been demonstrated in several different contexts and systems
(
Skinner et al., 2009; Wolins et al., 2005, 2006
). Second, the identity of the ER-
marker signal is also unambiguous. We showed that the signal for the ER marker
requires both transfection with an expression plasmid and the primary antibody.
Also, this transfection protocol results in varying levels of antigen in different cells,
and this is reflected in ER-marker signal levels. Finally, DAPI staining makes the
expression difference evident by identifying individual cells. The blue arrow points
to the DAPI-labeled nucleus with no surrounding signal in the ER-marker channel,
thus showing that the ER signal is transfection dependent. Note that the ER marker is
a fat synthesis enzyme (DGAT1) which increases TAG storage in lipid droplets and
the untransfected cell is almost devoid of perinuclear perilipin 3 coated fat droplets.
Also notable, the extranuclear DAPI staining represents small particles of condensed
DNA from the transfection. Revealing the cytoskeleton by staining cellular filaments
brings context to the signal of interest. Staining actin filaments with phalloidin-fluo-
rochrome conjugates defines the cell boundary, is simple, and is not antibody based.
Imaging is often used to localize proteins to compartments by comparing the sig-
nal of the protein of interest to specific organellar markers. Digital imaging allows
the precise overlap of images by showing each image as a different color. The
merged image in
Fig. 12.1
shows perilipin 3 puncta along the DGAT1-labeled ER
as previously reported (
Skinner et al., 2009
). Signal overlap is shown as a third color.
For example, red and green in an RBG image is shown in yellow (
Fig. 12.1
). In
Fig. 12.1
, the separate images for perilipin 3 and DGAT1 are patently dissimilar.
Nonetheless, copious yellow is seen in the merged view. This is due to the ER
and fat droplets overlapping in the thick perinuclear region of the cell, but not
due to colocalization of the signals. This example illustrates that caution must be
used when interpreting overlapping signals.
The goal of imaging is to present data as pictures. When visually scanning and
positioning cells for photography, it is important to observe the more stable
fluorochromes, usually the antibody-fluorochrome conjugates. This avoids
