Chemical Analysis | Protocol | Additional Information
||A golden-yellow crystalline powder.
||A bright-yellow powder.
||20ml of a 5% w/v aqueous solution should be clean, and almost free from suspended matter, and should have not than a very slight odor.
|pH of a 1% solution:
||Between 4.0 and 5.5 at 25 degrees Celsius.
||The spectral characteristics of Fluoro-Gold vary w/pH.
||A 0.1% solution in water has an excitation maximum of 414 nM and an emission maximum of 541 nM.
Fluoro-Gold bound to membranes at a physiological pH of 7.4 has an excitation band of 350-385nM and an emission band of 530-600nM.
||Not more than 0.35%.
||Not more than 0.1%.
||Less than 0.05%.
||Not more than 0.1%.
||Not more than 10 p.p.m.
||Less than 10 p.p.m.
||Not more than 30 p.p.m.
||Less than 10 p.p.m.
|Loss on drying:
||Not more than 1.0% after 3 hrs in vacuo at 60 degrees Celsius.
||Between 95.0 and 105.0% calculated w/reference to the dried material.
CAUTION: This is a drug for investigational use only in laboratory research animals or for tests in vitro. NOT FOR USE IN HUMANS. This drug should be used only by persons regularly engaged in conducting neuroanatomical studies and tests in vitro or in animals used only for laboratory research.
FLUORO-GOLD HISTOLOGICAL STAINING PROTOCOL:
- 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 compatibility with other histochemical techniques. It is also more resistant to fading, brighter and more permanent than most other fluorescent tracers.
- Storage and Shelf Life: Dry Fluoro-Gold 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.
- Vehicle: Fluoro-Gold can be dissolved in distilled water or 0.9% saline.
- 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.
- Dye Administration:
- Pressure injection - this is probably the most frequently used mode of application. Volumes injected range from .05-1 ul, typically .1-.2 ul.
- Iontophoresis - discrete, small injection sites result from 4-10 second pulsed iotophoretic (+5 to +10ua/10min) application.
- Crystal - a crystal of the tracer can be administered from the tip of a micro-pipette.
- Post-Operative Survival Periiod 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: Although any fixative, or no fixative, can be used, neutral phosphate buffered saline containing 4% formaldehyde (10% formalin) is most 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 (10um), frozen sections of fixed tissue (20-30um), and thin sections cut from tissue imbedded in either plastic (.2-4um) or paraffin (3-10um). 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 immunocytochemistsry, then sections are air-dried and directly coverslipped with DPX.
- Examination and Photography: Fluoro-Gold can be visualized with a fluorescence microscope using a wide band ultraviolet excitation filter (excitation - 323 nm, emission - 620 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). 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:
Vehicle: 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 1-2% 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 um. Optimal iontophoresis parameters are +1 to +5u amps delivered with pulsed current (4-10 seconds on, 4-10 seconds off) over a 10-20 min period.
Injection Sites: Virtually any central or peripheral nervous system structure can be injected with Fluoro-Gold for analysis of retrograde transport.
Transport and Survival Time: Fluoro-Gold is used as a retrograde axonal tracer, although orthograde axonal transport does occur. For retrograde transport, the survival times typically range from 1-14 days. A 2-4 day survival interval 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.
Tissue Processing: Tissue processing is covered in detail 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, its 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. Sections are typically mounted on gelatin-coated slides, air dried, immersed in xylene and coverslipped with DPX plastic mounting media (Sigma chemical Corp.). Tissue may also be viewed on slides without further processing.
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.