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荧光金 Fluoro-Gold 荧光金逆行标记大鼠视网膜神经节细胞 Fluorochrome Fluoro Gold
78000 荧光金试剂 Fluorochrome 授权代理,荧光金大量现货
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78000荧光金 Fluoro-Gold20mgfluorochrome

荧光金逆行标记大鼠视网膜神经节细胞

【摘要】 目的 将荧光金(flurogold,FG)注射于SD大鼠上丘,观察到被FG逆行标记的视网膜神经节细胞(retinal ganglion cells,RGC),以及SD大鼠RGC的数目及分布。方法 用3%FG双上丘注射,逆行标记RGC。5天后做视网膜定向铺片,在荧光显微镜下距离视乳头中心上下左右各2mm拍摄照片。利用计算机图像分析系统做RGC计数。结果 右眼RGC单位面积数量472.9±54.13,左眼RGC单位面积数量471.5±52.54。双眼RGC单位面积数量相比差异无显著性(P>0.05)。视网膜血管自视盘发出呈放射状,血管走行区无标记细胞。距视盘2mm始RGC分布较致密,视网膜周边部细胞分布较为稀疏。结论 用荧光金逆行标记上丘方法来标记RGC,是实验条件下研究RGC数量动态变化的可靠、有效方法。

  【关键词】 视网膜神经节细胞;荧光金

  Retinal ganglion cells retrograde labelled by injection of fluorogold

  LI Yue-hua,MA Ke,Xu Liang.

  Department of Ophthalmology,Beijing Chaoyang Hospital,Beijing 100020,China

  【Abstract】 Objective Retinal ganglion cells (RGC) were retrograde labeled by injection of 3% fluorogold into both side of superior colliculus to observe the numbers and distribution of RGC .Four photographs were taken from every quadrant of the retina 2mm from the center of optic disc. Retinal ganglion cells were quantitatively analyzed by computer.Methods To identify RGC,we applied the 3%fluorogold to the superior colliculi.The retinal were examined through a fluorescence microscope after 5 days,four photographs were taken from every quadrant of the retina 2mm from the center of optic disc. Retinal ganglion cells were quantitatively analyzed by computer.Results The numbers of RGC were not significantly different between two groups. The numbers of right eye was 472.9±54.13.The numbers of left eye was 471.5±52.54.Conclusion The method that retinal ganglion cells (RGC) retrograde labeled by injection of fluorogold is reliable and effective.

  【Key words】 retinal ganglion cells;fluorogold

视网膜神经节细胞(retinal ganglion cells,RGC)的慢性进行性丢失是青光眼zui主要的病理生理学特征。高眼压、缺血等原发性损伤首先引起一部分RGC的丢失,丢失的节细胞产生出许多毒性物质,使其周围RGC的生存环境恶化,细胞膜离子通透性增加,线粒体膜遭受损害,致使细胞进入凋亡程序,导致细胞丢失[1]。因此准确的RGC计数是研究青光眼发病机制及视神经保护治疗的关键指标。该实验将荧光金(flurogold,FG)注射于SD大鼠上丘,观察到被FG逆行标记的RGC,以及SD大鼠RGC的数目及分布。

  1 材料与方法

  1.1 试剂与器械 荧光金(荧光金公司,美国),脑立体定位仪900型(David Kopf仪器公司,美国),荧光显微镜(AH-2,奥林巴斯公司,日本),联想Mustek扫描仪。

  1.2 实验动物 Sprague-Dawley大鼠10只,购自北京维通利华实验动物技术有限公司。雄性,重量200~240g,裂隙灯检查大鼠前节,包括角膜、前房、虹膜和晶状体无异常改变。Mydrine散瞳,检眼镜观察眼底,包括视盘、眼底血管及部分视网膜无异常改变者。实验动物在12h光照/12h黑暗的条件下饲养。

  1.3 逆行标记 采用双上丘注射法逆行标记RGC。大鼠腹腔注射进行麻醉,将其固定在脑立体定位仪上,0.5%碘伏消毒皮肤,于正中切开皮肤向下分离达颅骨。牙科钻钻开颅骨相应部位,用微量注射器吸取3%FG(溶于0.9%生理盐水中),每侧上丘注射两点(前囟后5.9 和 6.4mm,旁开1.4mm,深4.0mm),每点注射1.5μl,留针5min。

  1.4 视网膜定向铺片 实验第5天,实验动物在深麻醉下,于上方12点处做球结膜缝线标记,立即取出眼球固定于4%多聚甲醛磷酸缓冲液中2h。沿角膜缘后0.5mm剪开眼球,去除角膜和晶状体,分离视网膜并将其定向平铺,空气中自然干燥,市售无色指甲油封片。

  1.5 荧光照片RGC计数 在荧光显微镜下使用V波段荧光激发,距视盘中心2mm上下左右各拍摄照片1张,照片放大倍数125×,分别代表上、下、左、右四个象限的RGC数量。

  1.6 图像分析 使用联想Mustek扫描仪将照片以100dpi扫入计算机,使用彩色颗粒分析软件(CPAS)进行节细胞计数,将上、下、左、右4张照片节细胞数量累加,代表该眼节细胞单位面积数量。

  1.7 统计学方法 使用SPSS for Windows 10.0统计软件包,采用配对t检验,P<0.05差异具有显著性,P<0.01差异具有非常显著性。

  2 结果

  2.1 FG标记的RGC的形态 在FG标记后5天,大鼠视网膜铺片观察到标记的RGC细胞质中荧光均匀,边界清晰,有些细胞可见有荧光充盈的细胞突起,细胞外无荧光染料渗漏(见图1)。
  2.2 RGC计数及分布双眼RGC单位面积数量 见表1。两眼相比较差异无显著性(P>0.05)。视网膜血管自视盘发出呈放射状,血管走行区无标记细胞。距视盘2mm始RGC分布较致密,视网膜周边部细胞分布较为稀疏(见图2)。但细胞内荧光仍较明显。
  表1 双眼RGC单位面积数量 略
  3 讨论

  利用轴浆流逆向输送的特点,在颅内RGC投射的核团上丘、外侧膝状体[2]、视神经断端近眼球一侧放置辣根过氧化物酶或Diamidido Yellow,FG(Fluorogold),Evan Blue,Fluoro-Ruby(FR)[ 2~5]等各种荧光物质,通过逆向轴浆流将这些物质带到RGC胞体内,产生特定的染色效果,标记出RGC,而RGC层中的其他细胞则不能着色。而HRP参与细胞代谢,不能在细胞内长期存留。某些示踪剂如快蓝、核黄等,易于从标记细胞内扩散到周围组织,且照射时褪色较快,即使保存在低温、避光条件下,仍不能长期保存。另外应用传统的组织化学技术无法将RGC与视网膜内其他神经元严格区分,尤其是与移位的无长突细胞鉴别。利用轴浆流逆向输送的特点,目前多用荧光金逆行标记上丘方法来标记RGC,是实验条件下研究RGC数量动态变化的可靠、有效方法。
  荧光金在紫外线照射下,其激发光波长323nm,发射光波长为408nm,标记的RGC呈金黄色强荧光,能标记细胞质,而细胞核不着色,能很好显示树突分支,细胞外无荧光染料渗漏,不易扩散,与周围组织分界清晰,除RGC层外,其余各层中均无荧光标记的细胞。Selles-Navarro I[6]等研究发现应用荧光金行上丘逆行标记RGC后,细胞质内荧光金的存在不超过3周,标记后12天、14天、21天与标记后37天有明显差异。随时间进展荧光金可能丢失荧光或被代谢[7]。该实验于动物处死前5天行荧光金逆行标记上丘,标记的RGC呈金黄色强荧光,各象限均匀。通过逆行标记RGC可以记录它的累积丢失,比通过凋亡标记(如TUNEL染色)更好,通过凋亡标记在一定的时间只能看到少量细胞[8]。此标记方法广泛应用于RGC的发生和凋亡[9]、视网膜缺血[6]、视神经横断、视神经再生的研究。
该实验的标记方法为两点法双侧上丘注射,因为有95%以上的大鼠RGC投射到上丘[10],10%左右的RGC的轴突投射到同侧外侧膝状体。因此,采用对侧上丘注射标记单眼RGC的方法并不可靠,至少有10%的RGC不被标记。上丘注射法还包括一点法[10]、三点法[11]。以往我们也采取过每侧上丘注射一点的标记方法,发现这种方法能够成功标记RGC的比率比较低,而且对注射精度要求高,注射点必须位于上丘中心(前囟后5.3mm中线外2mm,深4.5mm)。如果注射偏离上丘中心位置,将导致视网膜RGC标记的不均匀。改用双侧上丘两点注射法标记出的RGC分布均匀,对视网膜各象限RGC数的统计学检验差异无显著性。另外发现RGC数量从中心到视网膜周边并没有大幅度衰减。三点标记法耗时长、增加动物感染机会、损伤较两点法重。比较上丘一点注射法、两点注射法和多点注射法,认为两点注射法效率高,标记良好,有比较好的稳定性。
实验证明用荧光金逆行标记上丘方法来标记RGC,是实验条件下研究RGC数量动态变化的可靠、有效方法。  
【参考文献】
1 Schwartz M, Belkin M, Yoles E, et al. Potential treatment modalities for glaucomatous neuropathy: neuroprotection and neuroregeneration . J Glaucoma, 1996,5: 427-432.
2 Kondo Y, Takada M, Honda Y, et al .Bilateral projections of single retinal ganglion cells to the lateral geniculate nuclei and superior colliculi in the albino rat. Brain Res, 1993,608(2): 204-215.
3 Farid Ahmed AK, Dong K, Setsu T, et al. Correlation between different types of retinal ganglion cells and their projection pattern in the albino rat. Brain Res, 1996,706(1): 163-168.
4 Farid Ahmed AK,Dong K, Hanna-Georges FB, et al. Retrograde double-labeling study of retinal ganglion cells from the ipsilateral VLGN and SC in the albino rat. Neurosci Lett, 1998,244(1): 47-51.
5 Levkovitch-Verbin H, Harris-Cerruti C,Groner Y, et al. RGC death in mice after optic nerne crush injury: oxidative stress and neuroprotection. Invest Ophthalmol Vis Sci, 2000,41(13): 4169-4174.
6 Selles-Navarro I, Villegas-Perez MP, Salvador-Silva M, et al. Retinal ganglion cell death after different transient periods of pressure-induced ischemia and survival intervals. A quantitative in vivo study. Invest Ophthalmol Vis Sci, 1996,37(10): 2002-2014.
7 G mez Ram rez AM,Villegas-p rez MP,Salvador M,et al.Use the flrorescent tracer fluorogold to identify the motoneuron population of the abducens nucleus:a quantitative in vivo study . Invest Ophthalmol Vis Sci, 1995, 36: 687.
8 Garcia-Valenzuela E,Shareef S,Walsh J,et al.Programmed cell death of retinal ganglion cells during experimental glaucoma.Exp Eye Res, 1995,61: 33-44.
9 Lagreze WA, Knorle R, Bach M, et al. Memantine is neuroprotective in a rat model of pressure-induced retinal ischemia. Invest Ophthalmol Vis Sci, 1998, 39Devil: 1063-1066.
10 Levkovitch-Verbin H,Harris-Cerruti C,Groner Y,et al.RGC death in mice after optic nerve crush injury:oxidative stress and neuroprotection. Invest Ophthalmol Vis Sci, 2000,41(13): 4169-4174.
11 Yoles E,Muller S,Schwartz M. NMDA-Receptor antagonist protects neurons from secondary degeneration after partial optic nerve crush.J Neurotrauma, 1997,14(9): 665-675.

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荧光金说明书


FLUOROCHROME,LLC 
1801 Williams Street, Suite 100
Denver, Colorado 80218 USA
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Fluoro-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  µ l, typically .1-.2  µ l.

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

QualityExpected ResultActual Result
AppearanceA golden-yellow, hygroscopic, crystalline powderA bright-yellow powder
OdorNoneNone
Solution20 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 odorPasses Test
pH of a 1% SolutionBetween 4.0 and 5.5 at 25 degrees Celsius4.6
Spectral characteristicsThe 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 nm

Fluoro-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

ChlorideNot more than 0.035%0.017%
SulfateNot more than 0.1%Less than 0.05%
Sulfated ashNot more than 0.1%Negligible
Heavy metalsNot more than 10 p.p.mLess than 10 p.p.m
SeleniumNot more than 30 p.p.mLess than 10 p.p.m.
Loss on dryingNot more than 1.0% after 3 hours in vacuo at 60 degrees Celsius0.1%
AssayBetween 95.0 and 105.0% calculated with reference to the dried material99.2%

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.

 

货号品名包装品牌
78000荧光金 Fluoro-Gold20mgfluorochrome

我们公司zui大优势是强大的采购,

1:基本什么都能进口,血清,抗体,耗材,还有部分限制进口的,

2 货品全,现经营过700多个品牌,基本所有生物试剂耗材都可以进口,特别是冷偏的产品那就更有优势,

3:提供加急服务,一般1-2周到货,超过时限加急费全免

4:价格公道,绝大部分价格有优势,当然不能保证100%产品都是价格zui低,因为价格zui低意味着没有服务.

5:良好的信誉,大部分客户我们提供货到付款服务,客户包括清华,北大 交大 复旦,中山等100多所大学,ROCHE,阿斯利康,国药 ,fisher等500多家公司

6:我们还是Santa,Advanced Biotechnologies Inc,fluorochrome,bangs,BBInternational,crystalchem,dianova,FD Neurotechnologies,Inc. FormuMax Scientific,Inc, Genebridege, Glycotope Biotechnology GmbH; iduron,Innovative Research of America, Ludger, neuroprobe,omicronbio, Polysciences,prospecbi, QA-BIO,quickzyme,RESEARCH DIETS,INC,sterlitech;sysy,TriLink BioTechnologies,Inc;worthington-biochem,zyagen等几十家国外公司授权代理。
7:我们还是invitrogen,qiagen,Midland BioProducts Corporationam,sigma;neb,roche,merck, rnd,BD, GE,pierce,BioLegend等知名大品牌批发,欢迎合作。

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产品相关关键字: 荧光金 Fluoro-Gold fluorochrome代理 fluorochrome总代 fluorochrome上海代理
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