本页总结了使用荧光乳胶微球或“珠子”作为逆行神经元示踪剂的大部分程序。
关于绿珠的特殊信息出现在本协议的末尾。
更多细节见：Katz, LC, Burkhalter, A. 和 Dreyer, WD 荧光乳胶微球作为体内和体外视觉皮层研究的逆行神经元标记。Nature 310: 498-500 (1984)，和 Katz, LC 和 Iarovici, DM 绿色荧光乳胶微球：一种新的逆行示踪剂。神经科学 34：511-520 (1990)。
供应方式： 随附的小瓶包含悬浮在蒸馏水中的浓缩珠溶液。如果红珠用于逆行追踪神经元通路，则溶液可以按原样使用或稀释使用。在大鼠视觉皮层中，使用红珠时，1:4 的稀释度似乎不会降低逆行标记的质量或程度。但是，对于初始实验，我们强烈建议使用溶液全强度。除蒸馏水外，标准盐溶液（NaCl、KCl）也可用作稀释剂。所提供的绿色珠子已完全准备好用于逆行追踪实验。不建议稀释绿色珠子。
储存：珠溶液应储存在加湿容器中，在冰箱中，以防止蒸发。不要冻结！被冻结的珠子将无法工作，也无法挽救。如果珠子变干，它们就不能被重组。这种材料的保质期尚未确定，但如果储存得当，它可以保持几年的良好状态。
应用：最好使用压力注射珠子（例如 1 毫升 Hamilton 注射器或加压空气注射系统）。对于局部电路工作，已通过具有 30-50 um 直径尖端的玻璃移液器注入非常少量 (30-50 nl)。对于常规逆行追踪，使用更大体积 (0.1-0.3 ul) 和更大直径的移液器吸头。然而，即使注射量很大，珠子也不会扩散到远离注射部位的地方（通常小于 1 毫米）。因此，为了标记投射到大型结构的所有或大部分神经元，应该进行几次注射。不建议将珠子的离子电渗应用作为传递示踪剂的有效方法。然而，珠子确实带有净负电荷。
生存时间：在大多数温血脊椎动物系统中，注射后的最短有效生存时间约为 24 小时。标记强度随着存活时间的延长而增加，最长可达 48 小时。48 小时后，未观察到标记强度增加（或减少）。这些值在冷血动物中可能有很大不同，建议初始生存时间为一周。最长存活时间尚未确定，但即使在 14 个月的存活时间后，标签的质量或范围也没有变化。单元格可能被永久标记。未观察到对动物或神经元的毒性作用。
固定和处理：标准固定是用 0.1 M 磷酸盐缓冲液洗涤，然后在 0.1 M 磷酸盐缓冲液 (pH 7.4) 中加入 4% 多聚甲醛。戊二醛会产生大量的组织自发荧光，这可能会掩盖珠标记的神经元，应尽可能避免。绿色珠子在戊二醛固定材料中将完全看不见。将冷冻切片收集在磷酸盐缓冲液中，安装在涂有明胶的载玻片上，然后风干。完全干燥后，可以将载玻片在二甲苯中清洗 1 分钟，然后用 Fluoromount 或 Krystalon 盖上盖子。Fluoromount 购自 Atomergic Chemetals Corp., Farmingdale, NY；来自新泽西州吉布斯敦的 Harleco (EM Industries) 的 Krystalon。
短暂接触酒精和二甲苯无害，但长时间接触（超过 5 分钟）会破坏珠子。珠子对甘油非常敏感，如果安装在含甘油的溶液中会迅速褪色。在需要使用非永久性透明/固定剂的情况下，水杨酸甲酯优于甘油。如果将载玻片保存在黑暗中，细胞中的标记至少一年不会褪色（尽管背景自发荧光可能会显着增加）。到目前为止，在塑料嵌入后保留珠子标记的尝试尚未成功。
观察：红色珠子中的染料是罗丹明，因此可以使用任何用于罗丹明的荧光滤光片。一些较旧的尼康罗丹明滤镜组会产生非常高的背景，这会使标记的细胞不可见。使用 Zeiss 和 Leitz 标准罗丹明滤光片都获得了良好的结果。对于绿色珠子，宽带荧光素过滤器效果很好。路西法黄的滤光片组提供更强烈的信号，但以更高的背景为代价。窄带荧光素滤光片会产生比宽带滤光片弱得多的信号。
即使经过长时间的观察或显微摄影，珠子也不会明显褪色。
使用低功率、低数值孔径的干式物镜（例如 X4、X10）通常看不到标记。如果细胞被强烈标记，X10 浸没物镜（数值孔径为 0.4 或更大）或更高功率的干物镜通常会显示细胞。然而，X25 浸没物镜通常会显示非常清晰标记的细胞，而低功率物镜会错过这些细胞。这些警告尤其适用于绿珠。在决定实验不起作用之前，请使用浸入物镜检查注射部位附近的部分。应该存在许多局部标记的细胞。
绿珠使用者的附加信息：在迄今为止所做的工作中，似乎年轻的动物比年长的动物更好地传递标签。此外，年轻动物的组织自发荧光较低。因此，如果可能的话，建议在涉及绿珠的实验中使用年轻的动物。
因为绿色珠子在比红色珠子更短的波长下被激发，所以组织自发荧光是一个更大的问题。因此，尽量减少自发荧光将产生更好的对比度信号。减少自发荧光的方法包括： 1) 使用更薄的切片（例如 30 um 而不是 40 或 50）；2) 使用较年轻的动物，和 3) 在盖玻片后立即检查切片（背景随着时间的推移而增加）。
This page summarizes most of the procedures for using fluorescent latex microspheres, or "beads" as a retrograde neuronal tracer.
Special information about green beads appears 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).
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 completely 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 um diameter tips. For routine retrograde tracing, larger volumes (0.1-0.3 ul) 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 approximately 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 completely 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 cover-slipping (background increases over time).