Main Protocol
Additional Information
Chemical Analysis
Main
Protocol
1. Background
The use of Fluoro-Gold is essentially the same as other fluorescent
tracers. The main difference is that Fluoro-Gold is more flexible in
terms of post-injection survival times, concentration range, tissue
treatment and compatability with other histochemical techniques.
2. Storage and Shelf Life
Dry Fluoro-Gold should be kept in the dark at 4 degrees Celsius,
although the dye will remain stable at room temperature for well over
six months. The dye in solution should be kept in the dark at 4 degrees
Celsius and will remain stable for at least six months.
3. Vehicle
Fluoro-Gold can be dissolved in distilled water or 0.9% saline, or
utilized as a suspension
in 0.2M neutral phosphate buffer.
4. Dye Concentration
Fluoro-Gold has been successfully used at concentrations ranging from
1-10%. Initially, a 4% concentration is advised. If undesirable necrosis
occurs at the injection site, or labeling is too intense, reduce the
concentration to a 2% solution.
5. Dye Administration
A. Pressure Injection - This is probably the most frequently used mode
of application. Volumes injected range from .05-1
µl, typically .1-.2
µl.
B. Iontophoresis - Discrete, small injection sites result from 4-10
second pulsed iontophoretic (+5 to +10ua/10min) application.
C. Crystal - A crystal of the tracer can be administered from the tip of
a micro-pipette.
6. Post-0perative Survival Period
Good retrograde labeling has been observed with periods ranging from two
days to two months. Survival periods of three to five days are typical.
Long survival periods enhance filling of distal processes without
diffusion of the dye from the cell.
7. Fixation
Most any fixative, or no fixative, can be used, Phosphate neutral
buffered saline containing 4% formaldehyde is frequently employed.
Fixatives containing high concentrations of heavy metals (e.g. osmium,
mercury) will quench the fluorescence, while high concentrations (over
1%) of glutaraldehyde may increase background fluorescence
8. Histochemical Processing
Tissue containing Fluoro-Gold may be processed according to virtually
any common histological technique. This includes cryostat sections of
unfixed tissue (10 µm), frozen sections of fixed tissue (20 µm), and
thin sections cut from tissue imbedded in either plastic (.2-4 µm) or
paraffin (3-10 µm). Frozen sections of fixed tissue are most frequently
used.
9. Combined Methods
At this point of processing, sections may be further processed for a
second marker such as autoradiography, HRP histochemistry,
immunocytochemistry, a second fluorescent tracer, fluorescent
counterstain, etc.
10. Mounting, Clearing and Coverslipping
Sections are typically mounted on gelatin-coated slides, air-dried,
immersed in xylene, and coverslipped with nonfluorescent DPX plastic
mounting media. Sections may be dehydrated with graded alcohols, unless
this is not compatible with a second tracer. If Fluoro-Gold is to be
combined with fluorescence immunocytochemistry, then sections are
air-dried and directly coverslipped with neutral buffered glycerine
(1:2).
11. Examination and Photography
Fluoro-Gold can be visualized with a fluorescence microscope using a
wide band ultraviolet excitation filter (Excitation - 331 nm, Emission -
418 nm at neutral pH). A gold color is emitted when tissue has been
processed with neutral pH buffer, whereas a blue color is emitted when
tissue is processed with acidic (e.g. pH 3.3) pH buffer. It can be
photographed digitally or with film (use Ektachrome 200-400 ASA film for
color prints and comparable speed film for black and white prints, for
example Tri-X). Most exposure times range from 10-60 second exposures,
depending on the objective magnification and the intensity of the label.
Thirty (30) second exposures are about average. Multiple exposures may
be exploited to simultaneously visualize Fluoro-Gold and another tracer.
Thus, UV would be combined with bright field illumination to
simultaneously locate Fluoro-Gold with HRP or silver grains in
autoradiography. Similarly, blue light excitation can be combined to
also visualize the green emission color of FITC, while green excitation
light may be used to simultaneously observe the red emission color of
propidium iodide, or ethidium bromide (a fluorescent counterstain).
Additional Information Concerning the
Use of Fluoro-Gold
V ehicle
For pressure injections through a microsyringe or micropipette,
Fluoro-Gold should be dissolved in distilled water or .9% saline.
Fluoro-Gold may also be utilized as a suspension in .2M neutral
phosphate buffer, however, the suspended particles may clog a fine
micropipette tip so distilled water or .9% saline is the preferred
vehicle. For iontophoresis, a 1% Fluoro-Gold solution is made up in .1M
acetate buffer (pH=3.3). Well-cleaned (95% ETOH, water) glass
micropipettes should have tips of 10-20 µm. Optimal iontophoresis
parameters are +1 to +5u amps delivered with pulsed current (4-10
seconds on, 4-10 seconds off) over a 10-20 minute period.
Injection Sites
Virtually any central or peripheral nervous system structure can be
injected with Fluoro-Gold for analysis of retrograde transport. In the
peripheral nervous system, ganglia and peripheral targets can be
studied. For studies of peripheral nerve, the nerve should be cut or
damaged and either dipped in, or injected with, aq 5% solution of
Fluoro-Gold. Since Fluoro-Gold is not significantly taken up by intact
fibers of passage, the fibers must be cut or severely damaged for uptake
of the dye to occur. It is not presently known if Fluoro-Gold can be
used for intracellular injections or used to study gap junctions,
however, some groups of investigators are presently testing these
possibilities.
Transport and Survival Time
Fluoro-Gold is used as a retrograde axonal tracer, although orthograde
axonal transport does occur. The survival time should be varied
(especially to very short survival times of 12 hours - 2 days) to
maximize orthograde transport in the specific neuronal system under
study. For retrograde transport, the survival times should be varied
from 4 days to 14 days. Seven to 10 days works for most systems,
although long pathways (e.g., spinal cord to brainstem) and pathways in
large mammals (e.g., cats, monkeys) may require longer survival times
(e.g., 14 days). In addition, since Fluoro-Gold remains fast within
retrogradely labeled neurons, survival times of several months will also
produce excellent results. For iontophoresis, 1 2-5 day survival time is
recommended.
Tissue Processing
Tissue processing is covered in detail in the use guide and in the
original publication (Schmued and Fallon, 1986, Brain Research
377:147-154). Since Fluoro-Gold is stable in many solvents and remains
fast within retrogradely labeled neurons, it's use is compatible with
many histochemical techniques. It can be used with other retrograde
tracers, immunofluorescence, PAP and ABC immunocytochemistry, HRP
histochemistry, autoradiography, counterstains (ethidium bromide is the
preferred fluorescent counterstain), paraffin embedding and plastic
embedding. However, if tissue is unfixed, additional processing of
tissue in aqueous solutions for over an hour or two will result in loss
of Fluoro-Gold fluorescence from labeled neurons. Fluoro-Gold may be
useful in electron microscopy. Fluoro-Gold can be used in a brain which
has been sectioned and transferred to phosphate buffer. Sections are
typically mounted on gelatin-coated slides, air dried, immersed in
xylene and coverslipped with DPX plastic mounting media (FLUKA Chemical
Corp., 255 Oser Avenue, Hauppauge, New York, 11788, Catalog #44581).
Tissue may also be viewed on slides without further processing, can be
run through graded alcohols for dehydration, or, for immunocytochemistry,
the sections can be air dried and directly coverslipped with neutral
buffered glycerine (1:2).
Examination and Photography
Fluoro-Gold is visualized with a fluorescence microscope using a wide
band ultraviolet (UV) excitation filter. Use the same filter pack you
would for other fluorescent retrograde tracers excited under wide band
UV (e.g., True Blue, Fast Blue, Nuclear Yellow), such as the Leitz Ploem
filter system A (Wide Band UV, Excitation filter BP 340-380), Mirror RKP
400, Barrier Filter LP 430). Objectives should be made especially for
fluorescence microscopy (such as that made by Zeiss) glycerine, or
water. Since plastic does absorb UV light, it is not advised to view
through plastic petri dishes, etc. Recommended films are T-Max (Kodak,
black & white) and Ektachrome 200 (Kodak, color slides). Exposure times
usually vary from 20 seconds to 1.5 minutes.
Chemical Analysis
|
