Fv1200

For atomic force microscopy (AFM) cells were fixed with 4% PFA in PBS and cell surface scans recorded using an MFP-3D atomic force microscope (Asylum Research), equipped with a silicon nitride cantilever (MLCT, k = 10 mN·m^− 1, Bruker AFM Probes). Images were recorded in contact mode in PBS at room temperature with a scan rate of 0.3 Hz. Image processing was performed using Gwyddion (http://gwyddion.net/) [41 ]. For ZO-1 and ROR2 immunofluorescence, samples were stained using an AlexaFluor488-labeled ZO-1 (#339188, Invitrogen) or ROR2 (#FAB20641G, R&D) antibody. Fluorescence was imaged on a confocal laser scanning microscope (Olympus, FV 1200 and Zeiss LSM800). Images were edited in ImageJ. For electron microscopy, the cells were fixed according to [42 (link)] for 24 h, then embedded in Epon post fixation with 1% OsO₄, 1.5% uranyl acetate, 1.5% tungstophosphoric acid followed by dehydration with ethanol. A diamond knife was used to prepare ultra-thin sections (50 nm). Micrographs were obtained using a Zeiss EM 912 electron microscope.

Mice were anesthetized with a mix of ketamine/xylacine and perfused with 4% paraformaldehyde in PBS. After an overnight postfixation, coronal vibratome sections (50 μm) were obtained, washed in PBS and PBS − 0.1% Triton X-100 (PBT) and incubated for 1 h at room temperature with 3-5% heat inactivated newborn calf serum in PBT. The primary antibodies used were α-Kdm1a (ab17721, 1:200 or 1:1000), α-NeuN (Chemicon MAB377, 1:500), α-GFAP (G3893, Sigma, 1:500), α-Cleaved Caspase-3 (9661, Cell Signaling, 1:500), α-Fos (226004, Synaptic Systems, 1:500), α-Parvalbumin (P3088, Sigma-Aldrich, 1:500), α-Pecam 1 (550274, BD Pharmingen™, 1:500), α-H3K27me3 (07-449, Millipore, 1:500), α-H3K27me3 (ab6002, Abcam, 1:250) and α-H3K27ac (ab4729, Abcam, 1:500). Nuclei were counterstained with a 1 nM DAPI solution (Invitrogen). For the TUNEL assay, we used the in situ Cell Death Detection Kit (11684795910, Roche) following the manufacturer’s instructions. DAB staining was performed according to the instructions of the product (11718096001, Roche). Images were taken with an Inverted Confocal Microscope Olympus FV1200 and Zeiss LSM 880.