Kiernan J A, Macpherson C M, Price A, Sun T
Department of Anatomy & Cell Biology, The University of Western Ontario, London, Canada.
Biotech Histochem. 1998 Sep;73(5):244-54. doi: 10.3109/10520299809141118.
Anionic dyes, notably acid fuchsine, strongly stain the nuclei and cytoplasm of neurons severely damaged by injury or disease. We provide detailed instructions for staining nervous tissue with toluidine blue and acid fuchsine for optimal demonstration of injured neurons. Degeneration was induced in the hippocampus of the mouse by systemic administration of kainic acid, and the resulting acidophilia was investigated using paraffin sections of the Carnoy- or Bouin-fixed brains. The affected cells were bright red with the toluidine blue-acid fuchsine sequence. Their nuclei were stainable also with alkaline Biebrich scarlet and with the 1,2-naphthoquinone-4-sulfonic acid-Ba(OH)2 method; all staining was blocked by benzil but was relatively refractory to deamination by HNO2. These properties indicated an arginine-rich protein. The nuclei were strongly acidophilic in the presence of a high concentration of DNA (strong Feulgen reaction), and acidophilia could not be induced in normal neuronal nuclei by chemical extraction of nucleic acids. The cytoplasmic acidophilia of degenerating hippocampal neurons was due to a protein rich in lysine (extinguished by alkalinity, easily prevented by deamination, and unaffected by benzil). Stainable RNA was absent from the perikarya of the affected cells, but normal neuronal cytoplasm did not become acidophilic after extraction of nucleic acids. We suggest that kainate-induced cell death is preceded by increased production of basic proteins, which become concentrated in the nucleus and perikaryon. Groups of small, darkly staining neurons were seen in the cerebral cortex in control and kainate-treated mice. These shrunken cells were purple with the toluidine blue-acid fuchsine stain, and were attributed to local injury incurred during removal of the unfixed brain.
阴离子染料，尤其是酸性品红，能强烈染色因损伤或疾病而严重受损的神经元的细胞核和细胞质。我们提供了用甲苯胺蓝和酸性品红染色神经组织的详细说明，以最佳显示受损神经元。通过全身注射 kainic 酸在小鼠海马中诱导变性，并使用 Carnoy 或 Bouin 固定脑的石蜡切片研究由此产生的嗜酸性。用甲苯胺蓝 - 酸性品红序列染色后，受影响的细胞呈鲜红色。它们的细胞核也能用碱性比布里希猩红和 1,2 - 萘醌 - 4 - 磺酸 - Ba(OH)₂ 法染色；所有染色均被联苯甲酰阻断，但对 HNO₂ 的脱氨基作用相对不敏感。这些特性表明存在富含精氨酸的蛋白质。在高浓度 DNA 存在下（强福尔根反应）细胞核呈强嗜酸性，通过化学提取核酸不能在正常神经元细胞核中诱导嗜酸性。变性海马神经元的细胞质嗜酸性是由于富含赖氨酸的蛋白质（被碱消除，易被脱氨基阻止，且不受联苯甲酰影响）。受影响细胞的胞体中不存在可染色的 RNA，但提取核酸后正常神经元细胞质不会变为嗜酸性。我们认为，kainate 诱导的细胞死亡之前是碱性蛋白质产量增加，这些蛋白质集中在细胞核和胞体中。在对照小鼠和 kainate 处理的小鼠的大脑皮层中可见成组的小的、深色染色的神经元。这些萎缩的细胞用甲苯胺蓝 - 酸性品红染色呈紫色，归因于在取出未固定的脑时发生的局部损伤。
