Rabbit anti pax6

Cells were fixed with paraformaldehyde (4% in PBS) for 20 min, followed by permeabilization (0.2% Triton X-100 in PBS for 10 min) and blocking (3% bovine serum albumin in 0.2% Triton X-100 in PBS for 10 min). Human iPSCs/NPCs were incubated in primary antibody for 2 h at room temperature (Mouse anti-polyQ (Millipore, MAB1574, 1:50), Mouse anti-OCT4 (Stem Cell Technologies, #60093, 1:200), Rabbit anti-PAX6 (Stem Cell Technologies, #60094, 1:300), Mouse anti-FUS (Abcam, #154141, 1:500), Rabbit anti-G3BP1 (MBL, #RN048PW, 1:500), Rabbit anti-DARPP32 (Abcam, #40801, 1:50), Rabbit anti-GABA (Sigma, #A2052, 1:100) and Chicken anti-MAP2 (Abcam, #5392, 1:500). Then, cells were 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 568F(ab’)2 Fragment of Goat Anti-Rabbit IgG (H+L) (ThermoFisher Scientific, #A-21069, 1:500)), Alexa Fluor 647 Donkey anti-Chicken (Jackson ImmunoResearch, #A-703-605-155, 1:500) and Hoechst 33342 (Life Technologies, #1656104) for 1 h at room temperature. PBS and distilled water wash were followed before the cover slips were mounted on Mowiol (Sigma, #324590).

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.