Ab183041

Immunofluorescence was performed as described42 (link) with minor modifications. Briefly, after washing, fixing and permealizing, cells were incubated overnight at 4 °C with primary antibodies: goat polyclonal Lamin B1 (sc-6216, Santa Cruz Biotechnology), mouse monoclonal Myc (60003-2-Ig, Proteintech), rabbit monoclonal MacroH2A1 (ab183041, Abcam), rabbit polyclonal H3K9me3 (A2360, Proteintech), rabbit monoclonal H3K27me3 (9733, CST) and rabbit monoclonal H3K4me3 (9751, CST). After incubation, cells were washed three times with blocking buffer (4% BSA in 1XPBS) and incubated two hours with secondary antibodies: Alexa Flour488 donkey anti-goat, Dylight 594 donkey anti-mouse, Dylight 594 donkey anti-rabbit (all from Jackson immunoResearch Lab). For biotinylated protein staining, cells were incubated with fluorescent Dye 647-Streptavidin (U0297, Abnova) for 2 hour at RT. After incubation with secondary antibodies or streptavidin, coverslips were washed three times and counterstained with 0.5 mg/ml DAPI (Beyotime Biotechnology) for 5 minutes at RT. Then coverslips were mounted in vectashield mounting medium (H-1000, Vector, Burlingame) and sealed on slides.

Protein samples were separated on SDS-PAGE and transferred to polyvinylidene difluoride membranes, and probed with primary antibodies: anti-Lamin B1 (sc-6216, Santa Cruz Biotechnology), anti-macroH2A1 (ab183041, Abcam), anti-LAP2 (14651-1-AP, Proteintech), anti-EMD (10351-1-AP, Proteintech), anti-MAN1 (sc-50458, Santa Cruz Biotechnology), anti-Lamin A/C (sc-6215, Santa Cruz Biotechnology); anti H3 (ab1791, abcam), anti H3K9me3 (A2360, Proteintech), anti H3K27me3 (9733, CST), anti-b-Actin (AGM11086, AOGMA) and anti-GAPDH (AGM12183, AOGMA). Then bound primary antibodies were detected with AffiniPure peroxidase–conjugated 2^nd antibodies (Jackson immunoResearch Lab). The blots were developed with ECL Plus Western Blotting Substrate (32132, Thermo Scientific), and imaged by the FluorChem M System (ProteinSimple, Santa Clara, CA, US).

Immunohistochemical analysis was performed using thick vibratome sections, as well as paraffin sections, by means of both fluorescent and immunoperoxidase detection. The spinal cord and brain sections were stained using antibodies to Nestin (R&D), SOX2 (BD Biosciences), βIII-tubulin (R&D), MAP2 (Sigma-Aldrich), GFAP (DAKO), NF-200 (Sigma-Aldrich,) macroH2A.1 (ab183041, Abcam), human-specific anti-mitochondria antibody (ab92824, Abcam), and STEM-121 (Y40410, TakaraBio), anti- CD3, CD31 and CD68 (Roche Diagnostics) (all as 5 μg/ml solutions in PBS). In the case of immunofluorescence detection with the use of vibratome sections, Alexa Fluor 488 goat anti-mouse IgG (H + L) and Alexa Fluor 633 goat anti-rabbit IgG (H + L) (all dilutions 1:400; Invitrogen, USA) were used as secondary antibodies, the fluorescence was detected with a Nikon A1 laser scanning confocal microscope (Nikon Co., Japan). Immunoperoxidase staining was performed on thin paraffin sections with a Benchmark ULTRA immunostainer (Ventana, USA) utilizing the manufacturer-recommended detection system.