Fluoro-Gold Fluorescent Retrograde Axonal Tracer

Fluoro-Gold is the most widely used and effective neuronal retrograde tracer in the world. Fluorogold,trade name Fluoro-Gold,is also referred to as hydroxystilbamidine.

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. If you need to use more precise measurements, the molecular weight of Fluoro-Gold is 532.6 daltons.

Dye Administration
A. Pressure Injection – This is probably the most frequently used mode of application. Volumes injected range from 0.05-1 µl, typically 0.1-0.2 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.

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.

Fixation
Almost 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.

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.

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.

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).

Examination and Photography
Fluoro-Gold can be visualized with a fluorescence microscope using a wide band ultraviolet excitation filter. 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).

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Background

The use of Fluoro-Gold is essentially the same as other fluorescent tracers. Fluoro-Gold is more flexible in terms of post-injection survival times, concentration range, tissue treatment and compatibility with other histochemical techniques.

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