α mouse alexafluor488

ALI cultures were fixed in 4% paraformaldehyde (Alfa Aesar, Heysham, UK) for 15 min at room temperature. Cells were blocked and permeabilized with 10% BSA, 5% NGS, 5% normal rabbit serum, 5% normal mouse serum in PBS (0.1% TritonX-100) for 2 h and rinsed in PBS. Cells were stained with mouse anti-β-tubulin (Sigma) at 1:200 in 2% BSA in PBS (0.1% TritonX-100) overnight at 4 °C. Cells were washed 3 times with PBS (0.1% TritonX-100) and stained with α-mouse AlexaFluor488 (ThermoFisher Scientific, Waltham, MA) at 1:200 in 5% BSA in PBS (0.1% TritonX-100) for 2 h at room temperature. Cells were washed 5 times with PBS (0.1% TritonX-100) and re-blocked with 10% mouse serum in PBS for 3 h. Mouse anti-dsRNA antibody (Scicons, Szriak, Hungary) was labeled with an AlexaFluor555 labeling kit (ThermoFisher), pre-absorbed at 1:200 in 5% BSA, 5% normal mouse serum in PBS for 1 h, and added to cells overnight at 4 °C. Cells were washed 3 times with PBS (0.1% TritonX-100) and nuclei subsequently stained with DAPI (ThermoFisher) at 1:50,000 for 20 min. Inserts were mounted using FluorSave (EMD Millipore) and imaged on a Leica TCS SP8 resonant scanning confocal microscope (Leica, Wetzlar, Germany) with Leica LasAF software.

The antibodies used in this study were α-CD44 (Dako, M708201), α-CD44-FITC (Miltenyi-Biotec, 130-098-210), α-CD133-APC (Miltenyi-Biotech, 130-098-829), α-phospho-histone H3 (Cell signaling, 9701), IgG1-FITC (Miltenyi-Biotec, 130-098-847), IgG1-APC (Miltenyi-Biotech, 130-098- 846), α-Olig2 (Millipore, ab9610), α-Mouse Alexa Fluor 488 (ThermoFisher Scientific, A11029), α-Mouse Alexa Fluor 568 (ThermoFisher Scientific, A11004), α-Rabbit Alexa Fluor 488 (ThermoFisher Scientific, A11008) and α-Rabbit Alexa Fluor 568 (ThermoFisher Scientific, A11011).

The following primary antibodies were used for Immunoblotting, ChIP or IF applications: α-JAZF1 (1:500, produced by Pineda, animal 1), α-ZNHIT1 (1:1000, HPA019043, Sigma-Aldrich, Soeborg, Denmark), α-GFP (1:1000, 11814460001, Sigma-Aldrich), α-H2A.Z (1:1000 or 5 µg/IP, 39944, Active motif, Carlsbad, CA, USA), α-H2A.Zac (1:1000 or 1 µg/IP, ABE1363, Merck Millipore, Darmstadt, Germany), α-alpha Tubulin (1:1000, 39527, Active motif), α-H4K5ac (1:1000, 39700, Active motif), α-H4K16ac (1:1000, 39167, Active motif), α-H2AK5ac (1:1000, 39108, Active motif), α-H3K14ac (1:1000, 39599, Active motif), α-H3 (1:1000, ab1791, Abcam, Cambridge, MA, USA), α-JAZF1 (1:100, HPA066967, Atlas Antibodies, Stockholm, Sweden), α-Fibrillarin (1:100, NB300-269, Novus Biologicals, Centennial, CO, USA), α-Coilin (gift from Grahmam Dellaire). The following secondary antibodies were used for immunoblotting or IF applications: α-rabbit-HRP (1:20000, 31460, Thermo Fisher Scientific), α-mouse-HRP (1:20000, 31430, Thermo Fisher Scientific), α-mouse-Alexa Fluor 488 (1:200, A-11017, Thermo Fisher Scientific), α-rabbit-Alexa Fluor 594 (1:200, A-11072, Thermo Fisher Scientific), α-rabbit-Alexa Fluor 488 (1:200, A-11070, Thermo Fisher Scientific), α-mouse-Alexa Fluor 594 (1:200, A-11020, Thermo Fisher Scientific).