Uchihara T, Nakamura A, Yamazaki M, Mori O
Department of Neuropathology, Tokyo Metropolitan Institute for Neuroscience, Japan.
Acta Neuropathol. 2001 Jun;101(6):535-9. doi: 10.1007/s004010000306.
Double immunofluorescence for paired helical filament (PHF)-tau (AT8) and ubiquitin, enhanced by catalyzed reporter deposition amplification, was combined with thiazin red (TR), a fluorochrome, which has an affinity to fibrillary structures such as neurofibrillary tangles (NFTs). After recording these triple-fluorescent images, sections were subjected to the Gallyas silver impregnation method, so that four different staining properties could be compared on the same structure. Among pyramidal neurons quantified in the hippocampus from six cases of Alzheimer's disease, 60.3% were positive for ubiquitin, and were consistently positive for TR. TR-positive neurons (77.1%) harbored fibrillary structures in the cytoplasm and were always positive for the Gallyas stain, which stained the largest number of legions (94.5%). AT8-positive neurons without fibrillary structure were negative for TR (11.6%, pretangle neurons). Some of the pretangle neurons were positive for the Gallyas stain even without fibrillary structures. Appearance of TR stain and ubiquitin in NFTs, but not in pretangle neurons, suggests that ubiquitin is integrated into tau-positive neurons after their transformation into NFTs. Because TR-positive NFTs sometimes lacked ubiquitin-like immunoreactivity, involvement of ubiquitin may not be an early event during NFT formation. This combined method is now found useful in determining how molecules other than tau are involved during the evolution from tau-positive neurons to NFTs in various neurological disorders characterized by the deposition of tau.
通过催化报告沉积扩增增强的配对螺旋丝（PHF）-tau（AT8）和泛素的双重免疫荧光与噻嗪红（TR）相结合，TR是一种对神经原纤维缠结（NFTs）等纤维状结构具有亲和力的荧光染料。记录这些三荧光图像后，将切片进行Gallyas银浸染法处理，以便在同一结构上比较四种不同的染色特性。在6例阿尔茨海默病患者海马中定量的锥体神经元中，60.3%的神经元泛素呈阳性，并且始终对TR呈阳性。TR阳性神经元（77.1%）在细胞质中含有纤维状结构，并且始终对Gallyas染色呈阳性，Gallyas染色显示的病灶数量最多（94.5%）。没有纤维状结构的AT8阳性神经元对TR呈阴性（11.6%，即前缠结神经元）。一些前缠结神经元即使没有纤维状结构，Gallyas染色也呈阳性。TR染色和泛素出现在NFTs中，而不出现在前缠结神经元中，这表明泛素在tau阳性神经元转化为NFTs后才整合进去。由于TR阳性的NFTs有时缺乏泛素样免疫反应性，泛素的参与可能不是NFT形成过程中的早期事件。现在发现这种联合方法有助于确定在以tau沉积为特征的各种神经疾病中，除tau之外的其他分子在从tau阳性神经元演变为NFTs的过程中是如何参与的。
