Page 217 - Fundamentals of Light Microscopy and Electronic Imaging

200 FLUORESCENCE MICROSCOPY

entire text of the exercise before beginning the work. Work in pairs to make the
exercise go smoothly.
In this exercise we will stain tissue culture cells with organelle-specific fluo-
rescent dyes to examine the morphology and abundance of organelle systems and
to compare the performance characteristics of different filter sets. Many, but not
all, of these dyes can be used to stain cells in the living state. Large well-spread
cells, such as COS-7, U2OS, CHO, LLCPK cells, or primary cultures of endothe-
lial cells, if available, are appropriate for this exercise. Prepare representative
sketches of the labeled cells. Keep a record of your observations, including the
time course of the staining response, the stability of fluorescence, and other rele-
vant information. For this exercise pick one or more of the following marker
dyes. Instructions for preparing dye solutions are given in Appendix II.

Dye Cell structure Filter Set
Acridine Orange Lysosomes Fluorescein
Bodipy-Ceramide Golgi Apparatus Fluorescein
DAPI, Hoechst 33258 DNA DAPI
DiOC 6 Mitochondria; endoplasmic reticulum Fluorescein
Ethidium Bromide DNA Rhodamine
Lyso-Tracker Green Lysosomes Fluorescein
Mito-Tracker Red Mitochondria Rhodamine
JC1 Mitochondria Rhodamine
Rhodamine Phalloidin Actin Stress Fibers Fluorescein

Procedure:
Transfer a cell-containing coverslip from a culture dish to a 35 mm dish contain-
ing HMEM (MEM culture medium plus 10 mM HEPES pH 7.2 and without
serum). Buffering with HEPES (10 mM, pH 7.2) is necessary when handling and
examining cultured cells in room air. Serum and phenol red should be omitted
because their fluorescence decreases contrast. Remove the culture medium, add 2
mL HMEM and tilt gently to wash the medium over the cells. Pull off the medium
with a Pasteur pipette and replace it with 2 mL HMEM containing a specific fluo-
rescent dye at the appropriate concentration. Disperse the dye by gently rocking
the culture dish. Incubate for the indicated time, retrieve and save the dye solution
for reuse at a later time, rinse the coverslip 3 with 1 mL HMEM to remove any
excess unbound dye, wipe off the back of the coverslip with a damp Kimwipe to
remove cells and medium, and prepare a Vaseline mount as described in Figure
10-9. Draw off extra medium from the edge of the coverslip chamber with a filter
paper and examine the preparations immediately. For each dye employed,
describe the degree of staining specificity for organelles and explain why a dye
might not appear to stain specifically. Also describe the intracellular abundance
and morphological pattern exhibited by each type of labeled organelle. Make
sketches and include scale bars on your drawings.