Biology Reference
In-Depth Information
controls for determining signal specificity and other practical considerations for op-
timizing image quality.
INTRODUCTION AND RATIONALE
The fat droplet is the main energy depot in many cells and is composed of neutral
lipid surrounded by a protein and phospholipid monolayer. Fat droplets have been
observed in liver (
Mottram, 1909
) and in muscle fibers (
Bullard, 1912
) for over a
century. Further, imaging of fat droplets has revealed their close association with
mitochondria (
Craig, Eglitis, & McConnell, 1963
), the endoplasmic reticulum
(ER) (
Ashworth, Race, & Mollen Hauer, 1959
), and peroxisomes (
Blanchette-
Mackie et al., 1995
).
Before the discovery of perilipin 1 as a fat droplet-specific protein 20 years ago
(
Greenberg et al., 1991
), the existence of specific fat coat proteins was not appreciated.
Investigations sparked by this discovery revealed that fat droplets in mammalian cells
have a dynamic protein coat that controls access to the fat stores. This coat includes
structural proteins, enzymes, and transport proteins (
Brasaemle, Dolios, Shapiro, &
Wang, 2004; Liu et al., 2004
). Due to the large size and distinct morphology of fat
droplets, fluorescence microscopy has played a key role in these studies. With in-
creased availability of antibodies, fluorescence stains, and fluorescing biologic mole-
cules, fluorescence microscopy will continue to be an important tool.
Here we discuss strategies using light microscopy to image fat droplets and the
proteins that package fat. In addition to staining protocols, we discuss considerations
for choosing cell lines and optimal physical formats in which to grow and stain cells.
We detail strategies to eliminate ambiguous signals and accomplish the simultaneous
imaging of multiple molecules. Finally, we discuss image collection strategies, im-
age presentation, and the importance of microscope choice for the imaging outcome.
12.1
MATERIALS
1.
Twenty-two millimeter square glass coverslips (no. 1.5 thickness preferred),
glass slides, six-well tissue culture plates, finely pointed biological forceps
(tweezers) (see
Fig. 12.2
).
2.
Phosphate-buffered saline (PBS) at pH 7.2-7.4.
3.
Cell fixation solution: add 13.5 ml methanol stabilized 37% formaldehyde to
236.5 ml PBS to make a 2% solution.
4.
Microscopy buffer for cell permeabilization and antibody dilution: to about
400 ml PBS, add 5 g BSA, 0.5 g saponin, and 0.5 ml of a 20% w/v solution of
sodium azide, NaN
3
. Mix until dissolved and bring to 500 ml with PBS. Final
solution is 1% BSA, 0.1% saponin, and 0.02% sodium azide. Pass solution
through 0.2-
m
m filter. Microscopy buffer is stable for 2 months at room
temperature. If solution becomes cloudy from precipitation, replace.
