Bg af647

The procedure followed is equal to the one described in Li et al.^{18 (link)}. For convenience here the procedure is described also. Rinse 2x Coverslips containing Nup96-SNAP-tag cells (catalog no. 300444,CLS Cell Line Service) with warm PBS. In a 2.4% (w/v) formaldehyde(FA) in PBS solution for 40 s we preform prefixation before the samples were permeabilized in 0.4% (v/v)Triton X-100 in PBS for 3 min. Complete fixation was carried out in 2.4% (w/v) FA in PBS for 30 min followed by 3 Å~ 5 min washing steps in PBS after fixation. Quenching of FA was done by placing the samples in 100 mM of NH4Cl in PBS for 5 min and afterward washed 3x in PBS for 5 min each. Then, the sample was incubated for 30 min with Image-iT FX Signal Enhancer (catalog no. I36933, Thermo Fisher Scientific) and then stained with SNAP dye buffer (3 μM BG-AF647 (catalog no. S9136S, New England Biolabs) and 3 μM dithiothreitol in 0.5% (w/v) bovine serum albumin (BSA) in PBS) for 2 h at room temperature. We removed unbound dye by washing the coverslips 3x for 5 min in PBS. Samples were then mounted into custom sample holders in imaging buffers (50 mM of Tris/HCl pH 8, 10 mM of NaCl, 10% (w/v) d-glucose, 500 μg ml⁻¹ of glucose oxidase, 40 μg ml⁻¹ of glucosecatalase and 35 mM of MEA in H₂O). We sealed the holder with parafilm.