von Philipsborn Anne C, Lang Susanne, Bernard André, Loeschinger Jürgen, David Christian, Lehnert Dirk, Bastmeyer Martin, Bonhoeffer Friedrich
Max-Planck-Institut für Entwicklungsbiologie, Spemannstrasse 35, 72076 Tübingen, Germany.
Nat Protoc. 2006;1(3):1322-8. doi: 10.1038/nprot.2006.251.
Microcontact printing (microCP) of proteins has been successfully used for patterning surfaces in various contexts. Here we describe a simple 'lift-off' method to print precise patterns of axon guidance molecules, which are used as substrate for growing chick retinal ganglion cell (RGC) axons. Briefly, the etched pattern of a silicon master is transferred to a protein-coated silicone cuboid (made from polydimethylsiloxane, PDMS), which is then used as a stamp on a glass coverslip. RGC explants are placed adjacent to the pattern and cultured overnight. Fluorescent labeling of the printed proteins allows the quantitative analysis of the interaction of axons and growth cones with single protein dots and of the overall outgrowth and guidance rate in variously designed patterns. Patterned substrates can be produced in 3-4 h and are stable for up to one week at 4 degrees C; the entire protocol can be completed in 3 d.
蛋白质的微接触印刷（microCP）已成功用于在各种情况下对表面进行图案化处理。在此，我们描述一种简单的“剥离”方法，用于印刷轴突导向分子的精确图案，这些分子用作培养鸡视网膜神经节细胞（RGC）轴突的底物。简而言之，硅母版的蚀刻图案被转移到涂有蛋白质的硅酮长方体（由聚二甲基硅氧烷，PDMS制成）上，然后将其用作盖玻片上的印章。将RGC外植体放置在图案附近并培养过夜。对印刷蛋白质进行荧光标记，可以定量分析轴突和生长锥与单个蛋白质点的相互作用，以及在各种设计图案中的总体生长和导向率。图案化底物可在3-4小时内制备完成，在4℃下可稳定保存长达一周；整个实验方案可在3天内完成。
