Coverslips were prepared as previously described (14). Briefly, 24 mm by 50 mm no. 1 coverslips (Fisherbrand) were sonicated in a bath sonicator (Kendall) for 20 min with isopropanol, Milli-Q water, and 5 M KOH, with Milli-Q water rinses between each step. The coverslips were then sonicated for 5 min in methanol and transferred to a solution of 2-ml N-(2-aminoethyl)-3-aminopropyltrimethoxysilane (97%) (A0700, UCT Specialties), 10-ml glacial acetic acid, and 200-ml methanol. The coverslips were incubated in the silane mixture for 10 min, sonicated for 1 min, and then incubated for another 10 min. They were then rinsed with Milli-Q water and dried with nitrogen. To passivate the coverslips, 100 mg of maleimide polyethylene glycol (PEG) succinimidyl carboxymethyl ester (molecular weight, 5000; A5003-1, JenKem Technology) was dissolved in 1 ml of 100 mM phosphate buffer (pH 7.0). Two coverslips were sandwiched with 100 μl of the PEG solution in between for 1 hour at room temperature and protected from light. The coverslips were then washed with Milli-Q water and dried before being incubated overnight with 100 μl of 3 mM Halo ligand thiol (P6761 Promega or AcmeBiosciences Inc.) in 100 mM phosphate buffer (pH 7.0). Afterward, the coverslips were washed with Milli-Q water, dried, and stored in vacuum-sealed bags at −20°C.

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