Wouterlood F G, Jorritsma-Byham B
Department of Anatomy, Vrije Universiteit, Amsterdam, Netherlands.
J Neurosci Methods. 1993 Jun;48(1-2):75-87. doi: 10.1016/s0165-0270(05)80009-3.
We investigated the properties of biotinylated dextran-amine (BDA) as a neuroanatomical tracer at the electron microscopic level and we compared the results with those obtained previously with another tracer, the lectin Phaseolus vulgaris-leucoagglutinin (PHA-L). BDA was injected into various brain areas of rats. Following survival and fixation, vibratome sections were cut, subjected to a freeze-thaw treatment, and incubated overnight with avidin-biotin complex (ABC). Following reaction with diaminobenzidine (DAB)-hydrogen peroxide, the sections were processed for electron microscopy. In the electron microscope we observed that the reaction product occurred in the cytoplasm of cell bodies and in the matrices of dendrites, axons and axon terminals following ABC histochemistry of BDA-containing brain sections. The ultrastructural details of BDA-labelled neurones were generally better preserved than in PHA-L-labelled material, whereas at the same time penetration of the reagent into the sections was complete (incomplete in sections of PHA-L material). We conclude that the use of BDA as a neuroanatomical tracer in electron microscopy is a good substitute for PHA-L. The detection method for transported BDA is much faster and less complicated than that for PHA-L, while the results are better; that is, there is improved penetration coinciding with good preservation of ultrastructure. Keeping the limitations of BDA as a neuroanatomical tracer in mind, e.g., retrograde transport into local collaterals of axons that intermingle with anterogradely labelled axons, BDA seems well suited as a neuroanatomical tracer for electron microscopy.
我们在电子显微镜水平研究了生物素化葡聚糖胺（BDA）作为神经解剖学示踪剂的特性，并将结果与先前使用另一种示踪剂——菜豆白细胞凝集素（PHA-L）所获得的结果进行了比较。将BDA注入大鼠的各个脑区。在存活和固定后，切取振动切片，进行冻融处理，并用抗生物素蛋白-生物素复合物（ABC）孵育过夜。与二氨基联苯胺（DAB）-过氧化氢反应后，对切片进行电子显微镜处理。在电子显微镜下，我们观察到在含有BDA的脑切片进行ABC组织化学后，反应产物出现在细胞体的细胞质以及树突、轴突和轴突终末的基质中。与PHA-L标记的材料相比，BDA标记神经元的超微结构细节通常保存得更好，而与此同时，试剂对切片的渗透是完全的（PHA-L材料的切片中则不完全）。我们得出结论，在电子显微镜中使用BDA作为神经解剖学示踪剂是PHA-L的良好替代品。运输的BDA的检测方法比PHA-L的检测方法更快且更简单，而结果更好；也就是说，在超微结构保存良好的同时，渗透性得到了改善。考虑到BDA作为神经解剖学示踪剂的局限性，例如逆行运输到与顺行标记轴突交织的轴突局部侧支中，BDA似乎非常适合作为电子显微镜的神经解剖学示踪剂。
