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Fig.1 Amygdala cells (40X) after Fluoro-Gold injection in the PVN. Antibody is at 1/50,000
Fig.2 High magnification view of Fluoro-Ruby labeled axons, terminals and vascular pericytes as seen in the rat striatum following tracerinjection into the contralateral substantia nigra.
52-9400 荧光金 Fluoro-Gold 20mg Fluorochrome 52-9400 荧光金 Fluoro-Gold 50mg Fluorochrome 52-9600 荧光金抗体 Antibody to Fluoro-Gold 0.1ml×1 Fluorochrome 52-9500 红色荧光金 Fluoro-Ruby 30mg Fluorochrome 52-9610 Ki067 Antibody-W 200ul Fluorochrome 52-9620 GFAP Antibody-W 200ul Fluorochrome 52-9630 BrdU Antibody-W 200ul Fluorochrome 荧光金Fluoro-Gold文献资料Tosolini A.P., Mohan R, Morris R., \"Targeting the full length of the motor end plates regions in the mouse forelimb increases the uptake of Fluoro-Gold into corresponding spinal cord motor neurons,” Frontiers in Neurology (2013) 1-10.Tosolini A.P., Mohan R., \"Spatial characterization of the motor neuron columns supplying the rat forelimb,” Neuroscience 200 (2012) 19-30.Bowyer, J.F. and Schmued, L., Fluoro-Ruby labeling prior to an amphetamine neurotoxic insult shows a definitive massive loss of dopaminergic terminals and axons in caudateputamen. Brain Res., 1075, (2006) 236-239.*Schmued, L.C., \"Anti-retrograde and retrograde neuroanatomical tract tracing with fluorescent compounds,” Neuroscience Protocols, 94-050-02 (1994) 1-15.Van Bockstaele, E.J., Wright, A.M., Cestari, D.M., and Pickel, V.M., \"Immunolabeling of retrogradely transported Fluoro-Gold: sensitivity and application to ultrastructural analysis of transmitter-specific mesolimbic circuitry,” Journal of Neuroscience Methods, 55 (1994) 65-78.*Bowyer, J.F., Gough, B., Broening, H.W., Newport, G.D., and Schmued, L., \"Fluoro-Gold and Pentamidine Inhibit the In Vitro and In Vivo Release of Dopamine in the Striatum of Rat,” The Journal of Pharmacology and Experimental Therapeutics, 266 (1993) 1066-1074.Balercia, G., Chen, S., and Bentivoglio, M., \"Electron microscopic analysis of fluorescent neuronal labeling after photoconversion,” Journal of Neuroscience Methods 45 (1992) 87-98. [Link]*Chang, H.T., Kuo, H., Whittaker, J.A., and Cooper, N.G.F., \"Light and electron microscopic analysis of projection neurons retrogradely labeled with Fluoro-Gold notes on the application of antibodies of Fluoro-Gold,” Journal of Neuroscience Methods, 35 (1990) 31-37.Schmued, L., \"Fluoro-Gold and 4-Acetamido-4′-isothiocyanostilbene-2,2’disulfonic Acid: Use of Substituted Stilbenes in Neuroanatomical Studies,” Methods in Neurosciences, 3 (1990) 317-330.Schmued, L., Kyriakidis, K., Heimer, L., In vivo anterograde and retrograde axonal transport of the fluorescent rhodamine-dextran-amine, Fluoro-Ruby, within the CNS. Brain Res, 526 (1990) 127-134.Schmued, L.C., Kyriakidis, K., Fallon, J.H., and Ribak, C.E., \"Neurons containing retrogradely transported Fluoro-Gold exhibit a variety of iysosomal profiles: a combined brightfield, fluorescence and electron microscopic study,” Journal of Neurocytology, 18 (1989) 333-343.*Pieribone, V.A., Aston-Jones, G., \"The iontophoretic application of Flouro-Gold for the study of the afferents to deep brain nuclei,” Brain Research 475 (1988) 259-271. [Link]Reep, R.L., Baccala, M.J., Booth, M.P., and Goodwin, G.S., \"Combined retrograde and anterograde tracing of neuronal connections: Fluoro-Gold and autoradiography,” Journal of Neuroscience Methods, 23 (1988) 1-5.*Schmued, L.C. and Fallon, J.H., \"Fluoro-Gold: a fluorescent retrograde axonal tracer with numerous unique properties,” Brain Research, 377 (1986) 147-154.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.荧光金说明书FLUOROCHROME,LLC1801 Williams Street, Suite 100Denver, Colorado 80218 USATelephone:(303) 394-1000 e-mail: info@Fluorochorome.comFax: (303) 321-1119 website: www.Fluorochrome.comFluoro-Gold Protocol and Use Guide
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 compatibility with other histochemical techniques. 2. Storage and Shelf Life Dry Fluoro-Gold should be kept in a light tight closed container at 4 degrees Celsius. Stored properly, Fluorogold should have a shelf life exceeding one year. The dye in solution should also be kept in a light tight closed container at 4 degrees Celsius and should 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. If you need to use more precise measurements, the molecular weight of Fluoro-Gold is 532.6 daltons. 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 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 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. 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 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% 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. 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, a 2-5 day survival time is recommended. It is estimated that transport occurs at about 2 cm per day for mammals; it is slower for cold-blooded animals. 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
Quality Expected Result Actual Result Appearance A golden-yellow, hygroscopic, crystalline powder A bright-yellow powder Odor None None Solution 20 ml of a 5% w/v aqueous solution should be clean, clear and almost free from suspended matter, and should have not more than a very slight odor Passes Test pH of a 1% Solution Between 4.0 and 5.5 at 25 degrees Celsius 4.6 Spectral characteristics The spectral characteristics of Fluoro-Gold vary with pH A 0.1% solution in distilled water has a pH of 4.5 and excitation peak of 414 nm and emission peak of 541 nmFluoro-Gold bound to membranes at a physiological pH of 7.4 has an excitation band of 350 to 395 nm and an emission band of 530 to 600 nm
Chloride Not more than 0.035% 0.017% Sulfate Not more than 0.1% Less than 0.05% Sulfated ash Not more than 0.1% Negligible Heavy metals Not more than 10 p.p.m Less than 10 p.p.m Selenium Not more than 30 p.p.m Less than 10 p.p.m. Loss on drying Not more than 1.0% after 3 hours in vacuo at 60 degrees Celsius 0.1% Assay Between 95.0 and 105.0% calculated with reference to the dried material 99.2%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.
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