For GFP visualization cells were grown on poly-L-lysine (Sigma Aldrich) coated chamber slides (ThermoFisher Scientific), fixed in 4% formaldehyde (FA) and nuclei were counterstained with Hoechst 33342 (ThermoFisher Scientific).
For RAD21 immunofluorescence analysis in mESCs, we let single cells adhere for 30 min on poly-L-lysine coated slides. Next, pre-extraction of the non-chromatin-associated RAD21 fraction was performed by incubation with 0.1% Triton X-100 in PBS for 1 min followed by fixation with 4% FA. Staining was performed with rabbitanti-RAD21 (Abcam, ab154769, 1:200) followed by incubation with goat anti-rabbit Alexa Fluor 647 (Abcam, 1:250). Nuclei were counterstained with 4’,6-Diamidino-2-Phenylindole (DAPI) (ThermoFisher Scientific). For NPCs, cells were grown on poly-L-lysine coated coverslips fixed in 4% FA and stained with mouse anti-NESTIN (BD biosciences, 611659, 1:200) and rabbit anti-GFAP (DAKO, Z033429-2, 1:100) antibodies, followed by incubation with goat anti-mouse Alexa Fluor 488 and goat anti-rabbit Alexa Fluor 568 antibodies (both ThermoFisher Scientific, 1:250). Nuclei were counterstained with DAPI. Prior to imaging all samples were mounted with FluorSave reagent (Merck). Fluorescent confocal images were captured on a Leica SP5 system (Leica, Wetzlar, Germany). All the immunofluorescence images were processed using ImageJ software (version 1.53c).
For RAD21 immunofluorescence analysis in mESCs, we let single cells adhere for 30 min on poly-L-lysine coated slides. Next, pre-extraction of the non-chromatin-associated RAD21 fraction was performed by incubation with 0.1% Triton X-100 in PBS for 1 min followed by fixation with 4% FA. Staining was performed with rabbitanti-RAD21 (Abcam, ab154769, 1:200) followed by incubation with goat anti-rabbit Alexa Fluor 647 (Abcam, 1:250). Nuclei were counterstained with 4’,6-Diamidino-2-Phenylindole (DAPI) (ThermoFisher Scientific). For NPCs, cells were grown on poly-L-lysine coated coverslips fixed in 4% FA and stained with mouse anti-NESTIN (BD biosciences, 611659, 1:200) and rabbit anti-GFAP (DAKO, Z033429-2, 1:100) antibodies, followed by incubation with goat anti-mouse Alexa Fluor 488 and goat anti-rabbit Alexa Fluor 568 antibodies (both ThermoFisher Scientific, 1:250). Nuclei were counterstained with DAPI. Prior to imaging all samples were mounted with FluorSave reagent (Merck). Fluorescent confocal images were captured on a Leica SP5 system (Leica, Wetzlar, Germany). All the immunofluorescence images were processed using ImageJ software (version 1.53c).