LumafluorRetroBeads——逆向示踪首先

RetroBeads™逆向示踪荧光微粒是Lumafluor 公司*的逆向示踪产品，且是目前weiyi证实有效的示踪微粒（该产品的有效性经数百篇从无脊椎动物到哺乳动物实验的研究论文证实），目前Lumafluor 公司的绿色和红色荧光示踪微粒产品只能从本公司采购。
  该示踪产品体内注射高度集中不易扩散，信号强，对活细胞或活体无毒，且存留时间大于一年，与大多顺向示踪剂、原位杂交检测技术、免疫组化技术兼容。

部分产品信息如下：

该说明书总结了大多数使用荧光胶乳微球（beads）作为神经元逆向示踪剂的操作流程，其中关于绿色beads的信息请在本说明书末尾查看。更为详尽的信息请参考Katz, L.C.的以下论文：

1. Katz, L.C., Burkhalter, A., and Dreyer, W.D. Fluorescent latex microspheres as a retrograde neuronal marker for in vivo and in vitro studies of visual cortex. Nature 310: 498-500 (1984)

2. Katz, L.C. and Iarovici, D.M. Green fluorescent latex microspheres: a new retrograde tracer. Neuroscience 34: 511-520 (1990)
如有疑问，请邮件咨询info@lumafluor.com 或咨询：919 801-6244或Lumafluor中国经销商：上海金畔生物科技有限公司。

LumafluorRetroBeads操作说明

一、包装与稀释：

Lumafluor 的beads包装于一支密封的小瓶内，内含的浓缩beads悬浮于蒸馏水中。如使用红色Beads作为神经通路的逆向示踪，其稀释方法推荐采用：大鼠视皮层内可采用1：4比例进行稀释而不减少Beads的荧光标记强度和质量。然而对于实验我们不推荐使用稀释的Beads而使用原液进行注射示踪。除蒸馏水外，常规的盐溶液如NaCl、KCl溶液也可作为稀释液。如使用绿色Beads，则强烈建议使用原液，无需稀释。

二、储存: 

    为避免蒸发，Bead溶液应存放于冰箱内4度冷藏，切忌勿冷冻！ 冷冻的Beads将失去效用，无法使用。而变干的beads同样也无法使用（无法重悬），该产品并无明显的使用期限，如按要求存放可保存数年。

三、注射：

    Beads使用压力注射，如1 mlHamilton微量注射器或气压注射系统，如需小区域微量注射(30-50 nl)，可采用末端30-50 um直径的玻璃电极注射，而常规的逆向示踪（注射量0.1-0.3 ul）可使用更大直径的玻璃电极。尽管如此，即使注入更大的剂量，Beads，也不会明显从注射位点扩散（通常扩散距离少于1mm），因此，为尽量*标记投射到一个较大神经核团的神经元，需使用多点注射。尽管Beads带有负电荷，但是不建议采用离子渗透法注入示踪Beads。

四、存活时间：

    在大多数恒温脊椎动物系统中，检测Beads的zui短有效时间是24小时，在48小时内标记强度随着体内注射时间而延长，48小时以后荧光强度基本保持恒定。而在冷血动物中，检测Beads的时间推荐为1周。目前并未检测Beads的zui长可检测期，然而在Beads的荧光强度和质量至少可保持在体内14个月以上不变，而细胞可能会被yongjiu标记。目前并未发现Beads在动物或神经元中表现出毒性作用的报道。

五、固定和处理：

    标准的固定方法是：0.1 M PBS冲洗或灌注后在4%的多聚甲醛（0.1 M PBS配制，pH 7.4）中固定，用戊二醛固定会产生大量的组织自发荧光，妨碍Beads标记的神经元，并且绿色Beads在戊二醛固定的组织中将*观察不到，因此应尽量避免采用。如采用冰冻切片，切片应用PBS漂洗后用明胶包被的载玻片贴片晾干，在*风干后，再用二甲苯透明1分钟，然后用荧光封片剂（Fluoromount或Krystalon）封片。Fluoromount可从Atomergic Chemetals Corp., （Farmingdale, NY）公司购买; Krystalon可从

Harleco (EM Industries，Gibbstown, NJ）购买。切片只可短期暴露在乙醇或二甲苯中，但是长时间暴露（大于5分钟）会损坏Beads。Beads 对甘油非常敏感，在甘油环境中荧光会迅速萃灭，因此切忌使用甘油类封片剂，如无法避免，还可采用水杨酸甲酯代替甘油作为封片剂。封片后的切片如存放于黑暗环境中，荧光Beads标记的细胞可保持一年不萃灭（但是切片的自身荧光背景会增加）。迄今为止，还没有成功采用塑胶材料包被Beads标记的组织的记录。

• 观察:

    红色Beads中的染料为罗丹明，因此所有与罗丹明匹配的荧光滤镜均可使用，部分老的尼康公司的罗丹明滤镜因背景较高，可能导致无法观察到荧光Beads，通常Zeiss 和Leitz的标准罗丹明滤镜可获得较好的观察效果。而大多绿色荧光滤镜均可较好地观察绿色Beads的荧光结果，设置为荧光黄的滤镜可获得较强的明亮荧光，但是同时也带来较高的背景干扰。较宽波段的荧光滤镜比窄波段的荧光滤镜可以获得更强的荧光信号。在长时间的观察和拍照下Beads的荧光也不会淬灭。

  在低倍数或低数值孔径物镜下(如 X4, X10)通常难以观察到Bads的荧光信号，只有细胞被显著标记，X10的油镜（数值孔径大于0.4）或者更大倍数的物镜才可观察到。通常情况下，X 25的油镜可以清晰地观察到低倍镜下无法观察到的标记细胞。物镜的选用对绿色荧光Beads尤其重要。在做出实验失败的决定前（在目标区域无法观察到标记细胞），可先用油镜仔细观察注射位点附近的细胞是否被标记，此处应该观察到大量的标记细胞。

对使用绿色荧光Beads标记的额外提醒：目前已发现绿色荧光Beads在年青动物比年老动物中标记更为理想的情况，此外，年青动物的组织自发荧光（背景）也更低，因此，假如可以的话，在使用绿色荧光Beads做逆向标记实验时请尽量使用年轻动物。

因为绿色荧光Beads的激发光段比红色荧光Beads短，因此组织自发荧光是个麻烦，因此，应采用减少自发荧光的步骤以获得更理想的实验结果，这些方法有：（1）使用更薄的切片，如30微米比40或50微米理想；（2）使用年青的动物；（3）封片后立即观察拍照（随着时间增加背景也会增加）。

！！！！！！！！！！！！！！！！！！！

    对使用色荧光Beads标记的额外提醒：目前已发现绿色荧光Beads在年青动物比年老动物中标记更为理想的情况，此外，年青动物的组织自发荧光（背景）也更低，因此，假如可以的话，在使用绿色荧光Beads做逆向标记实验时请尽量使用年轻动物。

    因为绿色荧光Beads的激发光段比红色荧光Beads短，因此组织自发荧光是个麻烦，因此，应采用减少自发荧光的步骤以获得更理想的实验结果，这些方法有：（1）使用更薄的切片，如30微米比40或50微米理想；（2）使用年青的动物；（3）封片后立即观察拍照（随着时间增加背景也会增加）。

Protocols for Use of Fluorescent Latex Microspheres （rev. 11/07）

This sheet summarizes most of the procedures for using fluorescent latex microspheres, or “beads” as a retrograde neuronal tracer. Special information about green beads is at the end of this protocol. Further details are presented in: Katz, L.C., Burkhalter, A., and Dreyer, W.D. Fluorescent latex microspheres as a retrograde neuronal marker for in vivo and in vitro studies of visual cortex. Nature 310: 498-500 （1984）, and Katz, L.C. and Iarovici, D.M. Green fluorescent latex microspheres: a new retrograde tracer. Neuroscience 34: 511-520 （1990）. Questions, problems, or comments concerning the use of this material can be directed to: info@lumafluor.com or phone 919 801-6244.

How supplied: The enclosed vial（s） contains a concentrated solution of beads suspended in distilled water. If red beads are being used for retrograde tracing of neuronal pathways, the solution can be used as is, or diluted. In rat visual cortex, dilutions of 1:4 do not appear to reduce the quality or extent of retrograde labeling when using red beads. However, for initial experiments we strongly recommend using the solution full strength. In addition to distilled water, standard salt solutions （NaCl, KCl） can be used as diluents. The green beads, as supplied, are compley prepared for retrograde tracing experiments. Dilution of green beads is not recommended.

Storage: The bead solution should be stored in a humidified container, in a refrigerator, to prevent evaporation. Do not freeze! Beads that have been frozen will not work, and cannot be rescued. If the beads dry out, they cannot be reconstituted. No shelf life has been established for this material, but, when properly stored, it remains good for several years.

Application: Beads are best injected using pressure （e.g. a 1 ml Hamilton syringe, or pressurized air injection system）. For local circuit work, very small volumes （30-50 nl） have been injected through glass pipettes with 30-50 mm diameter tips. For routine retrograde tracing, larger volumes （0.1-0.3 ml） and larger diameter pipette tips are used. However, even with large injections beads do not diffuse far from the injection site （usually less than 1 mm）. Thus in order to label all or most of the neurons projecting to a large structure, several injections should be made. Iontophoretic application of beads is not recommended as an effective means to deliver the tracer. However, the beads do have a net negative charge.

Survival times: The minimum effective post-injection survival time in most warm-blooded vertebrate systems is approximay 24 hours. Labeling intensity increases with longer survival, up to 48 hours. After 48 hours, no increase （or decrease） in labeling intensity is observed. These values may be considerably different in cold-blooded animals, and initial survival times of a week are recommended. The maximum survival time has not been established, but labeling is unchanged in either quality or extent even after 14 month survival times. Cells probably are permanently marked. No toxic effects on either animals or neurons have been observed.

Fixation and processing: Standard fixation is a 0.1 M phosphate buffer wash followed by 4% paraformaldehyde in 0.1 M phosphate buffer （pH 7.4）. Glutaraldehyde will produce substantial tissue autofluorescence which may obscure bead-labeled neurons, and should be avoided if possible. Green beads will be compley invisible in glutaraldehyde fixed material. Frozen sections are collected in phosphate buffer, mounted on gelatin-coated slides, and air-dried. After complete drying, slides can be cleared in xylene for 1 minute, and covers lipped with Fluoromount or Krystalon. Fluoromount is available from Atomergic Chemetals Corp., Farmingdale, NY; Krystalon from Harleco （EM Industries）,Gibbstown, NJ. Brief exposures to alcohols and xylenes are not harmful, but long exposures （over 5 minutes） will destroy the beads. Beads are very sensitive to glycerol, and will fade rapidly if mounted in glycerol-containing solutions. Methyl salicylate is preferable to glycerol in situations where non-permanent clearing/mounting agents are indicated. If slides are kept in the dark, the labelling in cells will not fade for at least one year （although background autofluorescence may increase substantially）. Thus far, attempts to retain bead labeling after plastic embedding have not been successful.

Observation: The dye in the red beads is rhodamine, thus any fluorescence filter set for rhodamine can be used. Some older Nikon rhodamine filter sets give a very high background, which can make labeled cells invisible. Good results have been obtained with both Zeiss and Leitz standard rhodamine filters. For green beads, a wide-band fluorescein filter works well. Filter sets for Lucifer yellow give a more intense signal, but at the expense of higher background. Narrow-band fluorescein filters will give a much weaker signal than a broad-band filter. Beads do not fade appreciably even after long periods of observation or photomicrography.

Labeling is usually not visible with low power, low numerical aperture dry objectives （e.g. X4, X10）. If cells are strongly labeled, a X10 immersion objective （numerical aperture of 0.4 or greater）, or higher power dry objectives, will usually reveal the cells. However, frequently a X25 immersion objective will reveal very clearly labeled cells that lower power objectives miss. These caveats are especially true for green beads. Before deciding that an experiment did not work, examine sections in the vicinity of the injection site with immersion objectives. Numerous locally labeled cells should be present.

Additional information for green bead users: In work that has been done so far, it appears that younger animals transport the label better than older animals. In addition, tissue autofluorescence is lower in the younger animals. Therefore, it is advisable to use younger animals, if possible, in experiments involving green beads.

Because the green beads are excited at shorter wavelengths than red beads, tissue autofluorescence is a greater problem. Therefore, efforts to minimize autofluorescence will produce a better contrast signal. Ways to reduce autofluorescence include: 1） using thinner sections （e.g. 30 um rather than 40 or 50） 2） using younger animals, and 3） examining sections promptly after coverslipping （background increases over time）.

Information

For Reliable, Robust Retrograde Transport, There's Only One Choice: RetroBeads™ from Lumafluor.

RetroBeads™ from Lumafluor–the original microspheres for retrograde tracing and the only microspheres proven effective where it counts: in your experiments. Green and Red fluorescent RetroBeads™ are available exclusively from Lumafluor.

Do not be misled by the unsubstantiated claims of other suppliers!
No matter what they call their products, only Retrobeads™ from Lumafluor have been proven effective by hundreds of published papers in systems ranging from invertebrates to primates, and everything in between.

Lumafluor Retrobeads™:

• Highly confined injections–superb for detailed connectivity studies.
• Persist indefiniy （> 1 year!） in living cells, nontoxic.
• Compatible with most other anterograde tracers, in situ hybridization, and immunohistochemsitry.

New!

Retrobeads™ IX: Retrobeads™ deliver bioactive agents （such as neurotrophins and neurotransmitter agonists/antagonists） to localized regions; retrograde transport allows determining which neurons were exposed to the agents [Riddle et al. Nature 378:189, 1995 and Quattrochi et al. Science 245:984, 1989].

Retrobeads™ IX are specially prepared to facilitate adsorption of proteins and other bioactive compounds. Retrobeads™ IX are also more effective tracers in primate systems than standard Retrobeads™.

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