Immunogold labeling allows localization of proteins at the electron microscopy (EM) level of resolution, and quantification of signals. The present paper summarizes methodological issues and experiences gained from studies on the distribution of synaptic and other neuron-specific proteins in cell cultures and brain tissues via a pre-embedding method. An optimal protocol includes careful determination of a fixation condition for any particular antibody, a well-planned tissue processing procedure, and a strict evaluation of the credibility of the labeling. Here, tips and caveats on different steps of the sample preparation protocol are illustrated with examples. A good starting condition for EM-compatible fixation and permeabilization is 4% paraformaldehyde in PBS for 30 min at room temperature, followed by 30 min incubation with 0.1% saponin. An optimal condition can then be readjusted for each particular antibody. Each lot of the secondary antibody (conjugated with a 1.4 nm small gold particle) needs to be evaluated against known standards for labeling efficiency. Silver enhancement is required to make the small gold visible, and quality of the silver-enhanced signals can be affected by subsequent steps of osmium tetroxide treatment, uranyl acetate en bloc staining, and by detergent or ethanol used to clean the diamond knife for cutting thin sections. Most importantly, verification of signals requires understanding of the protein of interest in order to validate for correct localization of antibodies at expected epitopes on particular organelles, and quantification of signals needs to take into consideration the penetration gradient of reagents and clumping of secondary antibodies.

中文翻译：

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细胞培养和大脑中神经元的预嵌入免疫金电子显微镜方案的优化

免疫金标记允许在电子显微镜 (EM) 分辨率水平上定位蛋白质，并量化信号。本文总结了通过预嵌入方法研究突触和其他神经元特异性蛋白质在细胞培养物和脑组织中的分布的方法问题和经验。最佳方案包括仔细确定任何特定抗体的固定条件、精心策划的组织处理程序以及对标记可信度的严格评估。在这里，示例说明了样品制备协议的不同步骤的提示和注意事项。EM 兼容的固定和透化的良好起始条件是 PBS 中的 4% 多聚甲醛在室温下 30 分钟，然后用 0.1% 皂苷孵育 30 分钟。然后可以针对每种特定抗体重新调整最佳条件。每批二抗（与 1.4 nm 小金颗粒偶联）都需要根据已知的标记效率标准进行评估。需要银增强以使小金可见，并且银增强信号的质量会受到后续步骤的四氧化锇处理、乙酸双氧铀整块染色以及用于清洁金刚石刀以切割薄片的洗涤剂或乙醇的影响部分。最重要的是，信号验证需要了解感兴趣的蛋白质，以验证抗体在特定细胞器的预期表位上的正确定位，并且信号的量化需要考虑试剂的渗透梯度和二抗的聚集。