Imaging of Neutral Lipids and Neutral Lipid Associated Proteins - Methods in Cell Biology - page 215

FIGURE 12.4
Fat droplets and associated mitochondria imaged in a live muscle fiber. Muscle fibers
from mice overexpressing perilipin 5 were collagenase dispersed for 1 h in 10 mg/ml
collagenase in complete cell growth media supplemented with 25 mM HEPES, then exposed
to BODIPY 493/503 and mitotracker for 3 h after which dyes were washed out. Labeled fibers
were spread on a glass slide and imaged using standard wide-field fluorescence microscopy.
For clearer presentation, the green fluorescing BODIPY 493/503 is shown as red.
protein detection generally utilizes a two-antibody detection system. Cells are
incubated with an antibody against the protein of interest (the primary antibody).
Then species specific antibody-fluorochrome conjugates are used to recognize the
primary antibody. Both primary and secondary antibodies can contribute signal
unrelated to the antigen—that is, background. For example, if there is no protein
of interest present, all of the signal is background.
Unlike immunoblotting, where the predicted antigen migration in electrophoresis
provides an indication whether a signal is specific or spurious, immunostaining does
not provide this information. Thus, immunofluorescence studies require rigorous
controls to show signals are specific to the protein of interest. Demonstrating an in-
creased signal in cells with overexpressed protein of interest (e.g., by transfection)
can provide convincing evidence that the signal detected is from the protein of in-
terest. However, it does not guarantee that the endogenous protein will be discernable
from background. Using a variety of antibodies raised against the protein of
interest is another strategy to authenticate signal. Ideally, antibodies raised in
different species can be used to detect different epitopes of the same protein. If these
controls are not possible, imaging data can be supported by immunoblotting of
subcellular fractions. Specifically, if imaging and cell fractionation show a specific
signal around fat droplets and in fat droplet fractions, respectively, this is good
evidence that both signals are indeed against the putative antigen ( Wolins, Rubin,
& Brasaemle, 2001 ). Further, if conditions that perturb the subcellular localization
of a protein reveal the same changes by both immunofluorescence microscopy