Mab360

Bright field images were taken using an Olympus CKX41 microscope. Images were captured with CellSens standard software. For immunofluorescent images astrocytes were seeded in 4- or 8-well chamber slides (Millipore Sigma, C6932) coated with PDL and 20 μg/mL laminin I (Culturex, 3400-010-02). Cells were fixed in 100% methanol at − 20 °C for 5 min. After brief PBS wash, cells were blocked in 3% BSA for 1 h followed by overnight primary antibody incubation at 4 °C. Primary antibodies were: mouse anti-GFAP (1:1000; EMD Millipore MAB360); rabbit anti-NFκB (1:1000, Cell Signaling #8242). After extensive washing, secondary antibodies were added for 1 h at room temperature (1:1000 dilution). Nuclei were counterstained with DAPI containing mounting media (ThermoFisher Scientific, P36962). Images were acquired on a Zeiss Axio Observer Z1. Image J software was used for quantification.

The protein concentration of samples was determined using the DC (detergent compatible) protein assay (Bio-Rad, #5000112). Samples were prepared in a total volume of 20 μL at 15 μg/μL in Laemmli loading buffer. Samples were run on 7.5% Mini-PROTEAN gels for cytoplasmic proteins and 12.5% Mini-PROTEAN gels for histones (Bio-Rad, #456025). Primary antibodies used: Rabbit anti-mH2A2 (Invitrogen, PA5-57437) at 1:250 dilution, mouse monoclonal anti-β-ACTIN (Sigma-Aldrich, A5441, lot # 127M4866V, clone AC-15) at 1:1000, rabbit anti-lamin A/C (Abcam, ab108595) at 1:1000, rabbit anti-OLIG2 (Millipore, AB9610) at 1:500, mouse anti-GFAP (Millipore, MAB360), rabbit anti-H3 (Cell Signaling Technology, #9715) at 1:500, rabbit anti-PDFGRA (Cell Signaling Technology, #3164) at 1:500, rabbit polyclonal anti-macroH2A1 (Millipore, ABE215) at 1:500. Secondary antibodies used: Goat anti-rabbit IgG H&L (HRP) (Abcam, #6721, lot # GR3192725-6) at 1:20,000 dilution, goat anti-mouse IgG H&L (HRP) (Abcam, #6789) at 1:2000 dilution.