Page 218 - Fundamentals of Light Microscopy and Electronic Imaging

EXAMINING FLUORESCENT MOLECULES IN LIVING CELLS 201

Microscope set-up. Examine the cells with a 40–100 oil immersion lens
using epi-illumination with the 100 W mercury arc lamp. The microscope should
also be fitted with phase-contrast or DIC optics so that structural features in the
cells can be examined in transmitted light without having to move the specimen
or change the lens.

Precautions regarding fixatives. If formaldehyde or glutaraldehyde is
used as a fixative, use ventilation during preparation to prevent fumes from
contacting the eyes or skin. Place all fixatives and dyes in a toxic waste container
for proper disposal afterwards. Be sure to consult an experienced cell biologist
for advice and supervision.

Specific details:

DiOC /ER, mitochondria: DiOC is a lipophilic dye that stains organelles
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according to the lipid composition of their membranes. Cells are stained with 2
mL of 2.5 g/mL DiOC /HMEM for 1–2 min. Remove the dye and rinse the cells
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2 with HMEM. Mount and examine. Can you distinguish the mitochondria
from the ER? Does the nucleus also stain? Why? At dilute concentrations of dye
(1/10 the indicated concentration) only the mitochondria are stained. Living cells
can be treated for 30–60 min and examined the same as fixed cells. With longer
labeling times or at higher concentrations of dye, the ER also becomes labeled
and is seen as a beautiful lacy network at the periphery of the cell. Make sketches
of the stained organelles.

Bodipy-ceramide/Golgi apparatus: Rinse a coverslip containing cells in
HMEM; fix with 5 mL 0.5% glutaraldehyde in HMEM for 5 min. Remove the
fixative and rinse cells with HMEM several times. Incubate for 30 min at 5°C in
5 M C -bodipy-ceramide/BSA complex in a refrigerator. Ceramide is a lipid
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that partitions to certain lipid environments in the cell and BSA serves as a carrier
for the lipid. Remove and save the ceramide, rinse the cells at 22°C with HMEM
containing 3.4 mg/mL delipidated BSA several times over 1 hr, and then mount
and examine the cells. (The BSA removes excess ceramide and improves the
specificity of labeling.) If things go well, you’re in for a treat! Make drawings of
the Golgi apparatus from several cells. Are there any indications of staining of
pre-Golgi or post-Golgi structures? As an alternative, label living cells and
observe the pathway of dye uptake and partitioning from the plasma membrane to
endosomes to the Golgi. Wash a coverslip 3 with HMEM. Place the coverslip
in 5 M ceramide/BSA for 15 min at 37°C, then replace the solution with
complete culture medium and incubate for an additional 30 min (for endosomes)
or 60 min (for Golgi) at 37°C.

Ethidium bromide/nucleus: After rinsing in HMEM, the cells are fixed in
4 mL 0.5% glutaraldehyde in HMEM for 15 min. Remove the fixative, dehydrate
the cells in 100% ethanol for 1 min, and rinse in HMEM. Add ethidium bromide
to 5 g/mL (alternatively, DAPI to 0.5 g/mL) in HMEM, being careful not to