Schneider Gasser Edith M, Straub Carolin J, Panzanelli Patrizia, Weinmann Oliver, Sassoè-Pognetto Marco, Fritschy Jean-Marc
Institute of Pharmacology and Toxicology, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland.
Nat Protoc. 2006;1(4):1887-97. doi: 10.1038/nprot.2006.265.
Elucidating the molecular organization of synapses is essential for understanding brain function and plasticity. Immunofluorescence, combined with various fluorescent probes, is a sensitive and versatile method for morphological studies. However, analysis of synaptic proteins in situ is limited by epitope-masking after tissue fixation. Furthermore, postsynaptic proteins (such as ionotropic receptors and scaffolding proteins) often require weaker fixation for optimal detection than most intracellular markers, thereby hindering simultaneous visualization of these molecules. We present three protocols, which are alternatives to perfusion fixation, to overcome these restrictions. Brief tissue fixation shortly after interruption of vital functions preserves morphology and antigenicity. Combined with specific neuronal markers, selective detection of gamma-aminobutyric acid A (GABA(A)) receptors and the scaffolding protein gephyrin in relation to identified inhibitory presynaptic terminals in the rodent brain is feasible by confocal laser scanning microscopy. The most sophisticated of these protocols can be associated with electrophysiology for correlative studies of synapse structure and function. These protocols require 2-3 consecutive days for completion.
阐明突触的分子组织对于理解大脑功能和可塑性至关重要。免疫荧光结合各种荧光探针，是一种用于形态学研究的灵敏且通用的方法。然而，组织固定后表位掩蔽限制了原位突触蛋白的分析。此外，与大多数细胞内标记物相比，突触后蛋白（如离子型受体和支架蛋白）通常需要较弱的固定条件以实现最佳检测，从而阻碍了这些分子的同时可视化。我们提出了三种替代灌注固定的方案，以克服这些限制。在生命功能中断后不久进行简短的组织固定可保留形态和抗原性。结合特定的神经元标记物，通过共聚焦激光扫描显微镜可以在啮齿动物大脑中选择性检测γ-氨基丁酸A（GABA(A)）受体和支架蛋白gephyrin与已识别的抑制性突触前终末的关系。这些方案中最复杂的可以与电生理学相结合，用于突触结构和功能的相关研究。这些方案需要连续2至3天完成。
