Optimizing conditions and avoiding pitfalls for prolonged axonal tracing with carbocyanine dyes in fixed rat spinal cords.
Optimizing conditions and avoiding pitfalls for prolonged axonal tracing with carbocyanine dyes in fixed rat spinal cords.
复制标题
优化固定大鼠脊髓中使用碳青染料进行长时间轴突追踪的条件并避免陷阱。
DOI：
10.1016/j.jneumeth.2005.12.025
复制
发表时间：
2006
期刊：
影响因子：
--
通讯作者：
Windebank,AnthonyJ
中科院分区：
文献类型：
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作者：
Chen,BingkunK;Miller,StevenM;Mantilla,CarlosB;Gross,Louann;Yaszemski,MichaelJ;Windebank,AnthonyJ
我们的特点是在死后的脊髓轴突标记的方法，使用硅橡胶盘持有针涂有二碘和二氧在头端和尾端的脊髓。我们优化了DiI和DiO示踪技术在不同条件下的固定剂浓度（1%与4%多聚甲醛，PF），在室温（RT）与37°C长达24周。镶嵌在硅橡胶圆盘中的晶体涂层针提供了一种新的染料应用方法。纵向切片共聚焦显微镜显示DiI和DiO标记轴突膜和髓鞘。DiI的扩散距离明显长于DiO。两种染料在37°C下的迁移距离均大于RT。在1%和4%PF固定之间未发现染料标记的显著差异。长时间孵育后，有证据表明，染料扩散通过水性介质，并产生圆周标记的脐带。在贴标部位周围放置蜡封可防止这种不连续贴标。标记髓鞘在延长距离到脊髓表明，染料可以迁移细胞之间的潜伏期延长。我们的数据表明，较高的温度促进染料扩散沿着轴突，并证明，谨慎地DiI和DiO可作为特定的示踪剂在同一脊髓。
We have characterized a method of labeling of axons in the post-mortem spinal cord using a silastic disc holding pins coated with DiI and DiO at the rostral and caudal ends of the cord. We optimized the DiI and DiO tracing techniques under different conditions of fixative concentration (1% versus 4% paraformaldehyde, PF), at room temperature (RT) versus 37°C for up to 24 weeks. Crystal coated pins embedded in a silastic disc provided a novel method of dye application. Confocal microscopy of longitudinal sections showed DiI and DiO labeled both the axonal membrane and myelin sheath. DiI diffused significantly longer distances than DiO. Both dyes migrated greater distances at 37°C compared with RT. No significant difference of dye labeling was found between 1% and 4% PF fixation. After prolonged incubation there was evidence that dye diffused through the aqueous medium and produced circumferential labeling of the cord. Placing a wax seal around the labeling site prevented this non-contiguous labeling. Labeling of myelin sheaths at extended distances into the cord suggested that dye could migrate between cells with prolonged incubation periods. Our data suggested that higher temperature facilitated dye diffusion along the axons, and demonstrated that with caution DiI and DiO could be used as specific tracers in the same spinal cords.