Friedenberger Manuela, Bode Marcus, Krusche Andreas, Schubert Walter
Molecular Pattern Recognition Research Group, Institute of Medical Neurobiology, Otto-von-Guericke-University Magdeburg, ZENIT Building, Magdeburg, Germany.
Nat Protoc. 2007;2(9):2285-94. doi: 10.1038/nprot.2007.320.
This protocol details sample preparation and measurement procedures for a fluorescence technology capable of colocalizing hundreds of different proteins in a cell or tissue section. The procedure relies on fixation of samples and on the use of dye-conjugated tag libraries. To colocalize proteins, a sample is placed on the microscope stage of an imaging system (toponome imaging system (TIS)) performing sequential cycles of tag-dye incubation, imaging and bleaching to generate images for each localization cycle. TIS overcomes the spectral limitations of traditional fluorescence microscopy. Image processing reveals toponome maps, uncovering the coexistence of proteins at a location (protein clusters). The approach provides direct insight into the topological organization of proteins on a proteomic scale for the first time. If, for example, two dyes are used per cycle, 18 proteins in 4 visual fields can be colocalized in 21 h. Parallel TIS procedures using more than two dyes per cycle enhance the throughput.
本方案详细介绍了一种荧光技术的样品制备和测量程序，该技术能够对细胞或组织切片中的数百种不同蛋白质进行共定位。该程序依赖于样品的固定以及使用染料偶联标签文库。为了对蛋白质进行共定位，将样品放置在成像系统（拓扑体成像系统（TIS））的显微镜载物台上，进行标签 - 染料孵育、成像和漂白的顺序循环，以生成每个定位循环的图像。TIS克服了传统荧光显微镜的光谱限制。图像处理揭示了拓扑体图谱，揭示了蛋白质在某一位置（蛋白质簇）的共存情况。该方法首次在蛋白质组规模上直接洞察了蛋白质的拓扑组织。例如，如果每个循环使用两种染料，则在4个视野中的18种蛋白质可以在21小时内进行共定位。每个循环使用两种以上染料的并行TIS程序可提高通量。
