Biology Reference
In-Depth Information
with the primary or secondary antibodies. BODIPY 493/503 is widely utilized, be-
cause it is inexpensive, has a narrow excitation/emission spectrum that is compat-
ible with most green channels, is very bright, and more photostable than many other
lipophilic fluorochromes. However, if channel availability precludes using BOD-
IPY 493/503, other lipophilic fluorochromes with different excitation/emission
spectra are available (
Liu et al., 2009
). Finally, like DAPI, these dyes are potent
and care must be taken to avoid the unintentional staining of other specimen-
containing coverslips.
12.2.5
Fluorescent fatty acids
Cells incorporate fatty acids into triacylglycerol (TAG); therefore, TAG-containing
droplets canbe labeledwith incorporated fluorescent fatty acids. Bright fat droplet stain-
ing usually can be accomplished by adding 5-50mg/ml of labeled fatty acids (20 mg/ml
stock dissolved in DMSO) to culture media and incubating for one or more hours.
When using serum-containingmedia, thoroughmixing sufficiently solubilizes low con-
centrations of fatty acids. BODIPY
®
12 carbon fatty acids are useful because they
are incorporated into TAG-containing droplets, are available with emission spectra
in multiple wavelengths, and are bright and reasonably photostable. In our experience,
these molecules can give a higher signal to noise ratio than lipophilic fluorochromes.
Further, lipophilic fluorochrome labeling intensity (see above) increases dramatically
with fat droplet size, while BODIPY
®
fatty acid labeling is less size dependent, which
can be advantageous when imaging adipocytes or other cells with both large and small
fat droplets. Care must be taken in the interpretation of images produced when using
these fluorescent fatty acids, since fatty acids have multiple fates; we have observed
the labeling of both the ER and mitochondria with various dye-conjugated fatty acids.
In addition, we have observed changes in emission spectra when these fatty acids are
used at high concentrations. Finally, BODIPY fatty acids are not naturally occurring
and hence, the fluorophore may affect both the rate of processing and metabolic fate
of the labeled fatty acid. Thus, their utility for kinetic and partitioning studies is limited.
In addition, fluorescing cholesteryl BODIPY has been used to distinguish cholesteryl
ester droplets from TAG droplets (
Hsieh et al., 2012
).
12.2.6
Testing for optical bleed through and cross-reactivity
Optic filter bleed through can occur when using simple stains such as BODIPY and
DAPI. Using the BODIPY 493/503 stain as an example, bleed through can be tested
for as follows: (1) obtain the maximal signal by staining the cells with the highest
concentration of BODIPY 493/503 that will be used then and (2) confirm the pres-
ence of a strong signal in the green channel and the absence of appreciable signal in
the red and blue channels.
Signal that is not dependent on the primary antibody for a given channel can re-
sult from light bleeding through from another channel, or from antibody cross-
reactivity. Cross-reactivity can include, a fluorochrome-labeled secondary antibody
