Ixon3 ultra du897 emccd

For fixed-cell STORM imaging, cells were fixed with 4% paraformaldehyde for 20 min and then washed with 100 mM glycine in Hanks’ balanced salt solution (HBSS) to quench the free paraformaldehyde. Cells were permeabilized and blocked in a permeabilization solution with 0.1% Triton X-100, 0.2% BSA, 5% goat serum and 0.01% sodium azide in HBSS. Cells were then incubated overnight at 4 °C with an anti-AKAP150 (MilliporeSigma, 07–210; EMD Millipore, 07–210) antibody at a 1:500 dilution, followed by a 1–2-h incubation with goat anti-rabbit Alexa 647-conjugated antibodies at a 1:1,000 dilution. Cells were then post-fixed again in 4% paraformaldehyde, quenched with 100 mM glycine in HBSS and washed with HBSS to prepare for imaging. Immediately before imaging, the medium was changed to STORM-compatible buffer (50 mM Tris-HCl (pH 8.0), 10 mM NaCl and 10% glucose) with glucose oxidase (560 mg ml⁻¹), catalase (170 mg ml⁻¹) and mercaptoethylamide (7.7 mg ml⁻¹). STORM images were obtained using a Nikon Ti total internal reflection fluorescence (TIRF) microscope with N-STORM, an Andor iXon3 Ultra DU 897 EMCCD and a ×100 oil-immersion TIRF objective. Photoactivation was driven by a Coherent 405-nm laser, while excitation was driven with a Coherent 647-nm laser. Puncta localization was performed using both Nikon Elements analysis software.