STORM Imaging of Fixed and Live Cells

Cells were plated onto PLB-coated Lab-Tek II imaging chambers (Nunc), and all experiments were performed at 37 °C, 5% CO₂ in tissue culture incubators. Cells were fixed with the addition of paraformaldehyde to a final concentration of 4% and incubated at 37 °C for 15 mins. Cells were then washed in DPBS with the serial addition and removal of 500 μL DPBS (5 time; 200 μL of liquid was always maintained within the well to prevent desiccation of the PLB). Cells were permeabilised, blocked and labelled as above. For live cell imaging, cells were resuspended in growth media, and added to wells containing PLBs that contained an equal volume of DPBS. Imaging proceeded at 37 °C, 5% CO₂. For single-colour STORM imaging, slides were immersed in 0.22 μm-filtered STORM imaging buffer (560 μg/mL glucose oxidase, 34 μg/mL catalase, 1% β-mercaptoethanol, 25 mM glucose, 5% glycerol (all from Sigma-Aldrich), and 25 mM HEPES/DPBS pH 8 (Thermo Fisher Scientific), refreshed regularly to maintain a low-oxygen environment for optimal fluorophore blinking. For two-colour STORM imaging, slides were immersed in 0.22 μm-filtered OxEA imaging buffer (50 mM β-MercaptoEthylamine hydrochloride (MEA, Sigma-Aldrich), 3% (v/v) OxyFluor™ (Oxyrase Inc.), 20% (v/v) sodium DL-lactate solution (Sigma-Aldrich) in DPBS, pH adjusted to 8–8.5 with NaOH), as previously published^{64 (link)}.