Fluoro-Gold Antibody Protocol

Hypothalamic cells along the 3rd left
ventricle (40X) after Flouro-Gold injection
in the PVN.  Antibody is at 1/50,000.
 

You will receive the Antibody to Fluoro-Gold in a small vial. It is a polyclonal antibody raised in rabbit. The vial will contain approximately 100µl of the antibody solution. It should be immediately frozen and stored in a cold (-20 degrees C), dark, dry environment. The freezer section, without the frost free feature, of a good commercial refrigerator should suffice. If properly and continuously frozen, the antibody solution can be stored up to one year.

The antibody solution should remain frozen until ready for use. The 100µl aliquot of the Antibody is at a dilution of 1/100. The Antibody can be further diluted 1/50,000 to 1/100,000 times in BSA diluent (50 mM KPBS, 0.4% Triton, 1% BSA, 1% NGS) to produce 50ml to 100ml of working titer. After preparation, the antibody solution can be stored for up to seven days in a cool (4 degrees C), dark and dry environment. The refrigerator section of a good commercial refrigerator should suffice. We do not recommend that the solution be used beyond seven days after preparation.

Use of the Antibody

Fluoro-Gold can be injected using several different methods, including pressure, iontophoretic and other applications developed by a variety of researchers. See Schmued and Fallon, Fluoro-Gold: "A fluorescent retrograde axonal tracer with numerous unique properties," Brain Research, 377 (1986) 147-154 as well as Pieribone and Aston-Jones, "The Iontophoretic Application of Fluoro-Gold for the study of afferents to deep brain nuclei," Brain Research, 475 (1988) 259-271. Many researchers have developed their own modified procedures. Use of the antibody should not be dependent upon the methodology used to employ Fluoro-Gold.

After the Fluoro-Gold has been injected, floating sections (we used thirty um sections from a rat perfused with 4% formaldehyde) are incubated with the Fluoro-Gold Antibody solution overnight at 4 degrees C. Sections are washed, then incubated in Biotinylated GAR (Vector Labs) at 1/1000 for 1 hour at room temperature and washed again. Sections are then incubated in Avidin/Biotin (Vector Labs) at 1/1000 for 1 hour, washed and transferred to Diaminobenzidine (.04%) and Nickel Chloride (2.5%) in 0.1 M NaAcetate with 0.06% H₂O₂ for six minutes. Sections are then washed, mounted, dried, dehydrated and cover slipped.

It has been our experience that if stored and prepared in the manner set out above, each vial of the antibody should treat 300 to 1000 thirty um sections of the albino rat brain (or similar sized animals) if you use the Vector elite kit. However, you should experiment with the concentration and procedures to determine which best fits your circumstance.

Notice: The original and only true Fluoro-Gold (Fluorogold) is produced by Fluorochrome, LLC and marketed by Fluorochrome, LLC and Histo-Chem Inc.

Fluoro-Gold (Fluorogold) is an exclusive product of Fluorochrome, LLC. It has been sold by Fluorochrome and widely used since 1985. Other companies are marketing a product they claim  is the same as or equivalent to Fluoro-Gold. In fact, the chemical structures of these compounds seem to be different from Fluoro-Gold. Certain physical properties of the compounds may be very different.

*CAUTION: Fluoro-Gold, Antibody to Fluoro-Gold and Fluoro-Ruby are for investigational use only in laboratory research animals or for tests in vitro. NOT FOR USE IN HUMANS. These drugs should be used only by persons regularly engaged in conducting neuroanatomical studies and tests in vitro or in animals used only for laboratory research.