Live-cell Confocal Imaging of IκBα and p65

Cells were plated onto 35 mm-glass-bottomed dishes (Greiner Bio-One) and incubated on the microscope stage at 37 °C in humidified 5% CO₂. Several Zeiss confocal microscopes were used (LSM Pascal, Exciter, 510meta, 710 or 780) with fluar × 40 numerical aperture (NA) 1.3 or plan-apochromat × 63 NA 1.4 objectives and appropriate excitation and emission wavelengths for the two fluorophores. Image capture was performed using the Zeiss software, either ‘Aim version 4.2 utilizing the Autofocus macro68 (link)' on the 5-series microscopes or ‘Zen 2010b SP1' on the 7-series microscopes. Quantification of IκBα-eGFP fluorescent signal of whole cells was performed using region of interest analysis in ‘Zen 2010b SP1'. The data were exported as mean fluorescence intensity. For quantification of p65-mCherry fluorescence, Cell Tracker (version 0.6)69 (link)70 (link) was used to estimate mean nuclear and whole-cell fluorescence level, which was expressed as a nuclear to total ratio.