Mouse anti nestin

Immunocytochemistry experiments were performed as we described in ref. ^{63 (link)}. Cells were fixed with paraformaldehyde (4% in PBS) for 30 min, followed by permeabilization (0.2 % Triton X-100 in PBS for 10 min) and blocking (3% BSA in 0.2% Triton X-100 in PBS for 10 min). Cells were incubated with primary antibody for 2 h at room temperature (Rabbit anti-H3K9me3 (Abcam, #8898, 1:500), Rabbit anti-PAX6 (Stem Cell Technologies, #60094, 1:300), Mouse anti-OCT4 (Stem Cell Technologies, #60093, 1:200), Mouse anti-Nestin (Stem Cell Technologies, #60091, 1:500), Rabbit anti-SOX1 (Stem Cell Technologies, #60095, 1:100), Mouse anti-MAP2 (Sigma, #1406, 1:200), Rabbit anti-H3K9me3 (Abcam, #8898, 1:500)). Cells were then washed with 0.2% Triton-X/PBS and incubated with secondary antibody Alexa Fluor 488 goat anti-mouse (ThermoFisher Scientific, A-11029, 1:500), Alexa Fluor 568 goat anti-rabbit (ThermoFisher Scientific, A-11011, 1:500), and 2 µg ml⁻¹ Hoechst 33342 (Life Technologies, #1656104) for 1 h at room temperature. 0.2% Triton-X/PBS and distilled water wash were followed before we mounted the cover slips.