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5. Antibodies against proteins of interest (primary antibodies) and antibody-
fluorochrome conjugates recognizing primary antibody species (secondary
antibodies). Aliquot antibodies and store aliquots at
80 C and when
20 or
thawed do not refreeze.
6. DAPI (4 0 ,6-diamidino-2-phenylindole) stock solution is 10 mg/ml dissolved
in water (store at 4 C) and BODIPY 493/503 stock is 1 mg/ml in DMSO (store
at
20 C).
7. Mounting media for adhering the cell-bearing coverslip to the glass slide. We
routinely make and use a polyvinyl alcohol based solution commonly called
elvanol ( Wessel, Voronina, & Brooks, 2004 ). Similar solutions can be purchased
and some include DAPI.
8. An epifluorescence-equipped microscope with a camera system and optic filters.
12.2 METHODS
12.2.1 General considerations
We describe a simple staining protocol, because time and manipulation degrade cel-
lular morphology. We recommend formalin fixation, because solvent fixation de-
grades fat droplet morphology ( DiDonato & Brasaemle, 2003 ). Since coverslips
are easily transferred using forceps, it is most convenient and economical to grow
cells on coverslips. We find that 22 mm square coverslips in six-well plates are use-
ful for most applications; this allows independent treatment for the cells in each well.
Using both glass coverslips and slides yields the best images. To maximize the
information gathered, we routinely co-stain with two antibodies made in different
species and DAPI (4 0 ,6-diamidino-2-phenylindole), or a single antibody together
with the lipophilic fluorochrome BODIPY 493/503 and DAPI. Minimize light expo-
sure because fluorochromes are subject to photobleaching. Finally, do not allow the
cell-bearing side of the coverslip to dry out.
12.2.2 Cell lines
The cell line chosen is often the difference between obtaining a clear, easily imaged
answer or an ambiguous result. For many imaging applications, cells having broad
flat processes lessen ambiguities caused by overlapping structures. For example, the
perilipin 3 puncta falling along the ER in Fig. 12.1 (red arrowheads) are evident, and
it is likely that this ER-perilipin 3 association continues into the thick perinuclear
region. However, overlapping signal obscures this relationship. Cells with large flat
processes include primary fibroblasts, 3T3 cells, COS7, and undifferentiated 3T3-L1
or OP9. Round cells include HEK293 cells and well-differentiated 3T3-L1 adipo-
cytes. In addition to morphology, antibody availability and transfectability are con-
siderations in choosing cell lines.
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