LumafluorRetroBeads——逆向示蹤首先

RetroBeads™逆向示蹤熒光微粒是Lumafluor 公司*的逆向示蹤產品，且是目前weiyi證實有效的示蹤微粒（該產品的有效性經數(shù)百篇從無脊椎動物到哺乳動物實驗的研究論文證實），目前Lumafluor 公司的綠色和紅色熒光示蹤微粒產品只能從本公司采購。
  該示蹤產品體內注射高度集中不易擴散，信號強，對活細胞或活體無毒，且存留時間大于一年，與大多順向示蹤劑、原位雜交檢測技術、免疫組化技術兼容。

部分產品信息如下：

該說明書總結了大多數(shù)使用熒光膠乳微球（beads）作為神經元逆向示蹤劑的操作流程，其中關于綠色beads的信息請在本說明書末尾查看。更為詳盡的信息請參考Katz, L.C.的以下論文：

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)
如有疑問，請郵件咨詢info@lumafluor.com 或咨詢：919 801-6244或Lumafluor中國經銷商：上海起發(fā)實驗試劑有限公司。

LumafluorRetroBeads操作說明

一、包裝與稀釋：

Lumafluor 的beads包裝于一支密封的小瓶內，內含的濃縮beads懸浮于蒸餾水中。如使用紅色Beads作為神經通路的逆向示蹤，其稀釋方法推薦采用：大鼠視皮層內可采用1：4比例進行稀釋而不減少Beads的熒光標記強度和質量。然而對于實驗我們不推薦使用稀釋的Beads而使用原液進行注射示蹤。除蒸餾水外，常規(guī)的鹽溶液如NaCl、KCl溶液也可作為稀釋液。如使用綠色Beads，則強烈建議使用原液，無需稀釋。

二、儲存: 

    為避免蒸發(fā)，Bead溶液應存放于冰箱內4度冷藏，切忌勿冷凍！ 冷凍的Beads將失去效用，無法使用。而變干的beads同樣也無法使用（無法重懸），該產品并無明顯的使用期限，如按要求存放可保存數(shù)年。

三、注射：

    Beads使用壓力注射，如1 mlHamilton微量注射器或氣壓注射系統(tǒng)，如需小區(qū)域微量注射(30-50 nl)，可采用末端30-50 um直徑的玻璃電極注射，而常規(guī)的逆向示蹤（注射量0.1-0.3 ul）可使用更大直徑的玻璃電極。盡管如此，即使注入更大的劑量，Beads，也不會明顯從注射位點擴散（通常擴散距離少于1mm），因此，為盡量*標記投射到一個較大神經核團的神經元，需使用多點注射。盡管Beads帶有負電荷，但是不建議采用離子滲透法注入示蹤Beads。

四、存活時間：

    在大多數(shù)恒溫脊椎動物系統(tǒng)中，檢測Beads的zui短有效時間是24小時，在48小時內標記強度隨著體內注射時間而延長，48小時以后熒光強度基本保持恒定。而在冷血動物中，檢測Beads的時間推薦為1周。目前并未檢測Beads的zui長可檢測期，然而在Beads的熒光強度和質量至少可保持在體內14個月以上不變，而細胞可能會被yongjiu標記。目前并未發(fā)現(xiàn)Beads在動物或神經元中表現(xiàn)出毒性作用的報道。

五、固定和處理：

    標準的固定方法是：0.1 M PBS沖洗或灌注后在4%的多聚甲醛（0.1 M PBS配制，pH 7.4）中固定，用戊二醛固定會產生大量的組織自發(fā)熒光，妨礙Beads標記的神經元，并且綠色Beads在戊二醛固定的組織中將*觀察不到，因此應盡量避免采用。如采用冰凍切片，切片應用PBS漂洗后用明膠包被的載玻片貼片晾干，在*風干后，再用二甲苯透明1分鐘，然后用熒光封片劑（Fluoromount或Krystalon）封片。Fluoromount可從Atomergic Chemetals Corp., （Farmingdale, NY）公司購買; Krystalon可從

Harleco (EM Industries，Gibbstown, NJ）購買。切片只可短期暴露在乙醇或二甲苯中，但是長時間暴露（大于5分鐘）會損壞Beads。Beads 對甘油非常敏感，在甘油環(huán)境中熒光會迅速萃滅，因此切忌使用甘油類封片劑，如無法避免，還可采用水楊酸甲酯代替甘油作為封片劑。封片后的切片如存放于黑暗環(huán)境中，熒光Beads標記的細胞可保持一年不萃滅（但是切片的自身熒光背景會增加）。迄今為止，還沒有成功采用塑膠材料包被Beads標記的組織的記錄。

• 觀察:

    紅色Beads中的染料為羅丹明，因此所有與羅丹明匹配的熒光濾鏡均可使用，部分老的尼康公司的羅丹明濾鏡因背景較高，可能導致無法觀察到熒光Beads，通常Zeiss 和Leitz的標準羅丹明濾鏡可獲得較好的觀察效果。而大多綠色熒光濾鏡均可較好地觀察綠色Beads的熒光結果，設置為熒光黃的濾鏡可獲得較強的明亮熒光，但是同時也帶來較高的背景干擾。較寬波段的熒光濾鏡比窄波段的熒光濾鏡可以獲得更強的熒光信號。在長時間的觀察和拍照下Beads的熒光也不會淬滅。

  在低倍數(shù)或低數(shù)值孔徑物鏡下(如 X4, X10)通常難以觀察到Bads的熒光信號，只有細胞被顯著標記，X10的油鏡（數(shù)值孔徑大于0.4）或者更大倍數(shù)的物鏡才可觀察到。通常情況下，X 25的油鏡可以清晰地觀察到低倍鏡下無法觀察到的標記細胞。物鏡的選用對綠色熒光Beads尤其重要。在做出實驗失敗的決定前（在目標區(qū)域無法觀察到標記細胞），可先用油鏡仔細觀察注射位點附近的細胞是否被標記，此處應該觀察到大量的標記細胞。

對使用綠色熒光Beads標記的額外提醒：目前已發(fā)現(xiàn)綠色熒光Beads在年青動物比年老動物中標記更為理想的情況，此外，年青動物的組織自發(fā)熒光（背景）也更低，因此，假如可以的話，在使用綠色熒光Beads做逆向標記實驗時請盡量使用年輕動物。

因為綠色熒光Beads的激發(fā)光段比紅色熒光Beads短，因此組織自發(fā)熒光是個麻煩，因此，應采用減少自發(fā)熒光的步驟以獲得更理想的實驗結果，這些方法有：（1）使用更薄的切片，如30微米比40或50微米理想；（2）使用年青的動物；（3）封片后立即觀察拍照（隨著時間增加背景也會增加）。

！！！！！！！！！！！！！！！！！！！

    對使用色熒光Beads標記的額外提醒：目前已發(fā)現(xiàn)綠色熒光Beads在年青動物比年老動物中標記更為理想的情況，此外，年青動物的組織自發(fā)熒光（背景）也更低，因此，假如可以的話，在使用綠色熒光Beads做逆向標記實驗時請盡量使用年輕動物。

    因為綠色熒光Beads的激發(fā)光段比紅色熒光Beads短，因此組織自發(fā)熒光是個麻煩，因此，應采用減少自發(fā)熒光的步驟以獲得更理想的實驗結果，這些方法有：（1）使用更薄的切片，如30微米比40或50微米理想；（2）使用年青的動物；（3）封片后立即觀察拍照（隨著時間增加背景也會增加）。

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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