霍乱毒素亚单位 B（重组）、生物素-XX 偶联物

•请确认您所用的示踪剂交联到蛋白质上，或具有用于固定的伯胺——酰肼、赖氨酸可固定葡聚糖或蛋白质偶联物皆可。
•使用醛类固定剂交联示踪剂上的胺基。
•注入更大量或更高浓度的示踪剂。示踪剂通常注射1-20％浓度（10 mg/mL或更高）。
•确认您使用正确的荧光滤光片进行检测。您可吸取少量未稀释的示踪剂储液滴一滴到载玻片上，然后在显微镜下使用您所需的滤光片观察测试。这可验证示踪剂荧光是否可以被检测到，以及荧光显微镜的滤光片是否工作正常。
•回顾组织固定和处理步骤，确认是否存在可能影响示踪剂的试剂或处理步骤。

麦胚凝集素和霍乱毒素偶联物可以用于逆向示踪。它们在某些应用中可能会有一些顺向示踪。选择指南可以在这里找到（https://www.thermofisher.com/us/en/home/life-science/cell-analysis/cell-tracing-tracking-and-morphology/neuronal-tracing/protein-conjugates.html）。

要考虑的因素有示踪对象的大小、给样方式（注射，直接上样到组织等），示踪对象是否需要固定。以下链接详细介绍了我们提供的各类神经元示踪剂的详细信息以及选择方法：

•神经元示踪（https://www.thermofisher.com/us/en/home/life-science/cell-analysis/cell-tracing-tracking-and-morphology/neuronal-tracing.html）
•选择示踪剂（https://www.thermofisher.com/us/en/home/references/molecular-probes-the-handbook/fluorescent-tracers-of-cell-morphology-and-fluid-flow/choosing-a-tracer.html）
•成像分析（http://assets.thermofisher.com/TFS-Assets/BID/Reference-Materials/bioprobes-50-journal.pdf）

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）。

Confirm that the tracer you are using crosslinks to proteins or has a primary amine for fixation-either a hydrazide, lysine fixable dextran, or a protein conjugate.
Use aldehyde-based fixatives to cross link the amines on the tracer.
Inject a larger amount or higher concentration of the tracer. Tracers are generally injected at 1-20% concentrations (10 mg/mL or higher).
Confirm that you are using the correct fluorescent filter for detection. You can perform a spot test by pipetting a small amount of the undiluted stock solution of the tracer onto a slide, then view under the filter you are using on your microscope. This will confirm if the tracer fluorescence can be detected and the fluorescent microscope filter is working properly.
Review tissue fixation and handling procedures to confirm if any reagents or processing procedures could be affecting the tracer.

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