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Microcontact printing of axon guidance molecules for generation of graded patterns

Abstract

Microcontact printing (μCP) 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 °C; the entire protocol can be completed in 3 d.

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Figure 1
Figure 2: Silicon masters and fluorescent protein patterns.

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Acknowledgements

We thank Amber Raja for helpful comments on the manuscript, and Volker Zibat for the scanning electron microscopy image. This work was supported by the DFG (Grant 134/14-1 to M.B. and F.B.) and the DFG Research Center for Functional Nanostructures (CFN, Project E2.3). A.P. receives a stipend from the German National Acadamic Foundation.

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Correspondence to Martin Bastmeyer or Friedrich Bonhoeffer.

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von Philipsborn, A., Lang, S., Bernard, A. et al. Microcontact printing of axon guidance molecules for generation of graded patterns. Nat Protoc 1, 1322–1328 (2006). https://doi.org/10.1038/nprot.2006.251

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