Chromeo 494

Fixation and staining of the cells for stimulated emission depletion (STED) microscopy have been described previously [23 (link)]. Incubation with the primary antibody was performed overnight at 4 °C. The following antibodies were used: anti-Ku70 (#ab 83501) (Abcam, Berlin, Germany) and anti-IKKγ/NEMO (#SAB1404591) (Sigma-Aldrich, Taufkirchen, Germany). The following secondary antibodies were used: anti-rabbit ATTO 647N (dilution 1:200) and anti-mouse Chromeo 494 (dilution 1:50) (Active Motif, La Hulpe, Belgium).

HEK293 cells were fixed using 4% paraformaldehyde for 10 min at RT, followed by 3 times washing with PBS. Incubation with the primary antibody was performed overnight at 4 °C. Primary antibody was removed; cells were washed 5 times with PBS and then incubated with fluorescence-labeled secondary antibody in blocking buffer. The following secondary antibodies were used: Anti-rabbit ATTO 647N, dilution 1:200, and anti-mouse Chromeo 494, dilution 1:50 (Active Motif, La Hulpe, Belgium). Incubation was carried out at RT for one hour followed by another 5 times of washing. Finally, cells were embedded in Mowiol, pH 8.4.