A simple and reproducible protocol of glass surface silanization for TIRF microscopy imaging.

We describe a simple and reproducible protocol for the preparation of microscope glass slides for in vitro motility assays that use total internal reflection fluorescence microscopy. The developed method utilizes trimethylchlorosilane (TMCS) as a silanizing reagent, which in the presence of imidazole as a catalyst and under optimized conditions enables reproducible preparation of high-quality hydrophobic glass surfaces. This method presents a simplification and improvement in reproducibility over the commonly applied protocol utilizing dichlorodimethylsilane (DDS) as a silanizing agent. We demonstrate the applicability of the new method by performing the analysis of the interactions of a molecular motor, kinesin-1 with microtubules.