von Bartheld C S, Cunningham D E, Rubel E W
Hearing Development Laboratories, University of Washington Medical School, Seattle 98195.
J Histochem Cytochem. 1990 May;38(5):725-33. doi: 10.1177/38.5.2185313.
The molecule 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) is a fluorescent dye which diffuses within cell membranes. The properties of DiI diffusion and fluorescence are maintained in aldehyde-fixed tissue, thereby allowing selective neuronal tracing post mortem. We describe three modifications of this tracing method. First, while DiL diffuses along neuronal membranes the tissue can be decalcified in EDTA at 37 degrees C. Tracing in decalcified tissue extends the possible application of the DiI technique to the investigation of neuronal tissue enclosed in bony structures. Second, we describe a protocol that allows sectioning of DiI-injected tissue on a cryostat with minimal subsequent spread of DiI in dried sections. Third, we demonstrate that DiI label of fluorescent neurons in cryosections as well as Vibratome sections can be photo-oxidated and converted to a stable diaminobenzidine reaction product. The photo-converted DiI label is electron dense and allows analysis of labeled cell bodies and processes at the electron microscopic level. DiI does not stay confined to the surface cell membrane in fixed tissue but reaches internal organelles, presumably via membranes of the endoplasmic reticulum, and concentrates in microsomal structures adjacent to mitochondria. Photoconversion of DiI label is compatible with gold immunocytochemistry. Long-term incubation and subsequent photoconversion of post-mortem DiI-labeled neurons provides remarkable tissue preservation at the ultrastructural level.
分子1,1'-二硬脂酰基-3,3,3',3'-四甲基吲哚羰花青高氯酸盐（DiI）是一种能在细胞膜内扩散的荧光染料。在醛固定组织中，DiI的扩散和荧光特性得以保留，从而能够在死后进行选择性神经元追踪。我们描述了这种追踪方法的三种改进。第一，虽然DiI沿着神经元膜扩散，但组织可在37摄氏度下用乙二胺四乙酸（EDTA）脱钙。在脱钙组织中进行追踪将DiI技术的可能应用扩展到对包埋在骨结构中的神经元组织的研究。第二，我们描述了一种方案，该方案允许在低温恒温器上对注射了DiI的组织进行切片，且在干燥切片中DiI随后的扩散最小。第三，我们证明，冷冻切片以及振动切片机切片中荧光神经元的DiI标记可以进行光氧化，并转化为稳定的二氨基联苯胺反应产物。光转化的DiI标记具有电子密度，能够在电子显微镜水平分析标记的细胞体和突起。在固定组织中，DiI并不局限于表面细胞膜，而是可能通过内质网的膜到达内部细胞器，并集中在线粒体附近的微粒体结构中。DiI标记的光转化与金免疫细胞化学兼容。对死后DiI标记的神经元进行长期孵育并随后进行光转化，可在超微结构水平上实现显著的组织保存。
