Loup F, Weinmann O, Yonekawa Y, Aguzzi A, Wieser H G, Fritschy J M
Institute of Pharmacology, University Hospital Zurich, Zurich, Switzerland.
J Histochem Cytochem. 1998 Oct;46(10):1129-39. doi: 10.1177/002215549804601005.
We designed a protocol to improve the immunohistochemical analysis of human brain structures, which overcomes the limited detection sensitivity, high background, and intense autofluorescence commonly associated with human tissue. This procedure was evaluated by using antibodies against major GABAA receptor subunits (alpha1, alpha2, alpha3, gamma2) in autopsy and surgical specimens. Tissue blocks were briefly fixed by immersion and pretreated with microwave irradiation in sodium citrate buffer. Immunoperoxidase staining revealed a marked enhancement of cell surface immunoreactivity and reduction of background in microwave-irradiated tissue, irrespective of its origin. For confocal laser scanning microscopy, immunofluorescence staining was optimized with the tyramide signal amplification (TSA) technique. This procedure not only dramatically increased the sensitivity for antigen detection but also totally suppressed autofluorescence, thus revealing the cellular and subcellular distribution of GABAA receptor subunits. A distinct neuron-specific expression pattern of the alpha-subunit variants was observed in cerebral cortex and hippocampal formation, along with widespread expression of the gamma2-subunit. Of particular interest was the prominent alpha2- and alpha3-subunit staining on the initial axon segment of pyramidal neurons. This protocol represents a major improvement for high-resolution studies of human brain tissue aimed at investigating morphological alterations underlying neurological diseases.
我们设计了一种方案来改进对人脑结构的免疫组织化学分析，该方案克服了通常与人体组织相关的检测灵敏度有限、背景高和自发荧光强的问题。通过在尸检和手术标本中使用针对主要GABAA受体亚基（α1、α2、α3、γ2）的抗体对该程序进行了评估。组织块通过浸入进行短暂固定，并用柠檬酸钠缓冲液中的微波辐射进行预处理。免疫过氧化物酶染色显示，无论组织来源如何，微波辐射组织中的细胞表面免疫反应性均显著增强，背景降低。对于共聚焦激光扫描显微镜，使用酪胺信号放大（TSA）技术优化了免疫荧光染色。该程序不仅显著提高了抗原检测的灵敏度，还完全抑制了自发荧光，从而揭示了GABAA受体亚基的细胞和亚细胞分布。在大脑皮层和海马结构中观察到α亚基变体独特的神经元特异性表达模式，同时γ2亚基广泛表达。特别值得关注的是在锥体神经元的初始轴突段上突出的α2和α3亚基染色。该方案代表了针对旨在研究神经疾病潜在形态学改变的人脑组织高分辨率研究的重大改进。
