Dextran, Fluorescein and Biotin, 10,000 MW, Anionic, Lysine Fixable (Mini-Emerald) - FAQs

如果您想看到最精细的结构，应使用低分子量葡聚糖偶联物，例如3,000 MW葡聚糖偶联物。

请确保您使用的是葡聚糖是可固定形式（即包含伯胺）。不包含伯胺的葡聚糖将无法固定。另一个原因可能是葡聚糖的浓度过低，其使用浓度可以增加到10 mg/mL。

如果您选择的示踪剂是亲脂性染料并且用甲醇固定，脂质会随着甲醇流失。如果您使用甲醇固定，可以选择能够共价结合神经元内的蛋白质的示踪剂。

由于这些染料插入脂质膜，任何对膜的破坏都将导致染料流失。这包括用Triton X-100之类的去垢剂或是甲醇之类的有机溶剂通透。通透是胞内抗体标记所必需的，但它会导致染料流失。相反，CFDA SE之类活性染料能够共价连接到细胞组分，因此可以在固定和通透之后更好的保留。

DiI是一种亲脂性染料，多数保留在细胞脂质内。当细胞经去垢剂通透或是用乙醇固定时，会去除掉脂质和染料。如果需要通透，可以使用CM-DiI标记，因为CM-DiI会共价结合膜里面的蛋白；部分信号会随着固定/通透丢失，但残留的信号仍足以被检测出。

我们没有测定右旋糖酐偶联物的净电荷。净电荷取决于标记右旋糖酐所用的荧光团和制备偶联物所采用的方法。我们根据所用的荧光团将右旋糖酐标记为中性的或阴性的，但是染料和右旋糖酐的净电荷可能并不总是相同。Alexa Fluor、Cascade Blue、荧光黄、荧光素和Oregon Green右旋糖苷本质上是阴性的，而大多数标记两性离子罗丹明 B、四甲基罗丹明和Texas Red染料的右旋糖酐基本上是中性的。

我们已使用过3000至70,000分子量右旋糖酐，但3000至10000分子量右旋糖酐最常用于神经元示踪。3000 分子量的右旋糖酐用于对精细神经传递进行更详细的跟踪、研究间隙连接，并且扩散更快；而10000MW右旋糖酐具有较慢的分散速度、较长的胞内存留时间，且不会不穿过间隙连接。

NeuroTrace BDA-10,000 神经元示踪试剂盒（货号N7167）手册有一个 使用10,000 MW lysine-fixable biotin dextran amine（BDA）进行注射操作及神经元跟踪的实验方案。该试验方案也适用于其他荧光标记的右旋糖酐。 请参阅第4和5页的表1a和1b（https://tools.thermofisher.com/content/sfs/manuals/mp07167.pdf）。

固体和结晶形式的DiI以及其他相关染料（货号D282，D3911，D7757，和D12731）有时与一个特定的神经元接触，通过膜侧向扩散染上整个细胞。此外，我们的NeuroTrace组织标记糊剂也可挑到针上，置于特定的神经元上。 有关我们的神经元细胞标记方法的比较结果请参阅下表。 产品：标记方式：标记强度：特征 神经元特异性抗体：针对神经细胞表达蛋白的一抗：与蛋白质表达量成比例：唯一的神经元特异性标记方式。 亲脂性神经元示踪剂：疏水性染料掺入到细胞中的脂质：由于细胞中含有大量的脂类，这种标记方式能提供最强的标记：能够跟踪整个样品的神经元。 荧光标记的右旋糖苷：通常右旋糖苷采用显微注射：所有的细胞质可能被标记，标记密度可能会更高：能够标记单个神经元，从而避免了荧光背景干扰。 膜电位指示剂：染料通过水性缓冲液上样到活细胞里：取决于周围电场引起的结构变化，或者去极化引起的染料流入：膜电位的变化在各种生理进程中发挥核心作用，包括神经脉冲波传播，肌肉收缩以及细胞信号传导过程。

详细信息请查阅此网页（https://www.thermofisher.com/us/en/home/life-science/cell-analysis/cell-tracing-tracking-and-morphology/neuronal-tracing.html）。

If you want to see the most detailed structure you should use the low molecular weight conjugated dextrans such as the 3,000 MW dextrans.

Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.

Ensure that the dextran you are using is the fixable form (i.e., contains a primary amine). Dextrans that do not contain a primary amine will not be fixed. Another factor could be that the concentration of the dextran is too low, and the concentration use can be increased up to 10 mg/mL.

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If the tracer you chose is a lipophilic dye and fix with methanol, the lipids are lost with the methanol. If you have to use methanol fixation then choose a tracer that will covalently bind to proteins in the neurons.

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Since these dyes insert into lipid membranes, any disruption of the membranes leads to loss of the dye. This includes permeabilization with detergents like Triton X-100 or organic solvents like methanol. Permeabilization is necessary for intracellular antibody labeling, leading to loss of the dye. Instead, a reactive dye such as CFDA SE should be used to allow for covalent attachment to cellular components, thus providing for better retention upon fixation and permeabilization.

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DiI is a lipophilic dye that resides mostly in lipids in the cell, when cells are permeabilized with detergent or fixed using alcohol this strips away the lipid and the dye. If permeabilization is required CM-DiI can be used because this binds covalently to proteins in the membrane; some signal is lost upon fixation/permeabilization, but enough signal should be retained to make detection possible.

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We do not determine the net charge of the dextran conjugates. The net charge depends on the fluorophore used to label the dextran and the method of preparing the conjugate. We label some dextrans as neutral or anionic based on the fluorophore used, however the net charge of the dextran may not always be the same as the dye. The Alexa Fluor, Cascade Blue, Lucifer Yellow, fluorescein, and Oregon Green dextrans are intrinsically anionic, whereas most of the dextrans labeled with the zwitterionic Rhodamine B, tetramethylrhodamine and Texas Red dyes are essentially neutral.

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Dextrans with molecular weights from 3,000 to 70,000 have been used, however the 3,000 and 10,000 MW dextrans are most commonly used for neuronal tracing. The 3,000 MW dextrans are used for more detailed tracing of fine neuronal projections, investigating gap junctions, and diffuse more quickly; while the 10,000 MW dextrans have slower distribution, longer cellular retention, and do not cross gap junctions.

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The NeuroTrace BDA-10,000 Neuronal Tracer Kit (Cat. No. N7167) manual has a good protocol for injection procedures and neuronal tracing using the10,000 MW lysine-fixable biotin dextran amine (BDA). This protocol could potentially be applied to other fluorescent dextrans.

Please review Tables 1a and 1b on pages 4 and 5 - https://tools.thermofisher.com/content/sfs/manuals/mp07167.pdf

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The solid and crystalline forms of DiI and other related dyes (Cat. Nos. D282, D3911, D7757, and D12731) are sometimes placed in contact with a specific neuron where it will travel down the cell by lateral diffusion via the membrane. Alternatively, our NeuroTrace Tissue Labeling Paste can be scooped onto a needle and placed onto particular neurons.

Please see the information below for a comparison of our neuronal cell labeling methods:
Product:Method of labeling: Labeling intensity: Features
Neuron-specific antibodies: Primary antibodies directed to proteins expressed in neuronal cells: Proportional to the amount of protein expressed: Provides the only neuronal specific labeling method
Lipophilic neuronal ytracers: Hydrophobic dyes are incorporated into lipids in the cell: This labeling method provides the most intense labeling becuase of the abundant amount of lipids: Allows tracing of neurons throughout the sample
Membrane potential indicators: Dyes are loaded into live cells in aqueous buffers: Depends on either changes in structures due to the electrical field they are in, or dye influx due to depolarization: Changes in membrane potential play a central role in physiological processes, including nerve-impulse propagation, muscle contraction, and cell signaling

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Please check out this web page (https://www.thermofisher.com/us/en/home/life-science/cell-analysis/cell-tracing-tracking-and-morphology/neuronal-tracing.html) for details.

Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.