Mouse anti oct4

The iPSC colonies were dissociated using TrypLE™ Select (Gibco Life Technologies) for 5 min, washed in DMEM:F12 and centrifuged at 300 g for 5 min before removing the supernatant and fixing with 3.7% PFA in PBS on ice for 10 min. The cells were washed again, followed by further centrifugation at 300 g for 5 min. After blocking and permeabilizing in 5% goat serum plus 0.3% Triton-x-100 in PBS for 30 min on ice, the cells were incubated with primary antibodies diluted in 5% goat serum/PBS for 30 min on ice. Following a further 2–3 washes, the cells were incubated with species-specific secondary antibodies conjugated to Alexa Fluor (Life Technologies) in the dark for 30 min on ice. For conjugated antibodies, only one antibody incubation step was required. The wash protocol was repeated and the cells were resuspended in 2% FBS/PBS and analysed by a BD Accuri™ C6 flow cytometer (BD Biosciences, Ann Arbor, MI, USA). The antibodies used included: mouse anti-OCT4 (STEMCELL Technologies), mouse anti-SSEA4 (STEMCELL Technologies), mouse anti-TRA-1-60 (Millipore), and mouse anti-TRA-1-81 (Millipore). Isotype controls included anti-IgG2b, anti-IgG3, and anti-IgM. All isotype control antibodies were obtained from Life Technologies.

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.