Goat anti sox1

After the 14 d assay treatment of cells, they were fixed with 1% paraformaldehyde (Wako) for 30 min at room temperature. After fixation, they were washed with phosphate-buffered saline (PBS; Wako) and permeabilized with 0.2% Triton X-100 (MP Biomedicals, Santa Ana, CA)/1× Blocking One (Nacalai tesque, Kyoto, Japan)/PBS (PBS-B-T buffer) for 30 min at room temperature. Then, cells were incubated with primary antibodies (goat anti-SOX1 [as a neural progenitor marker, 1:1000; R&D Systems, Minneapolis, MN], rat anti-GFAP [as an astrocyte marker, 1:1000; Life Technologies], and mouse anti-MAP2 [as a neuron marker, 1:1000; Sigma-Aldrich]) in PBS-B-T buffer overnight at 4 °C. After washing with 0.1% Tween-20 (Wako)/PBS, cells were treated with secondary antibodies (anti-goat IgG [H+L] Alexa Fluor 488 [1:500; Life Technologies], anti-rat IgG [H+L] Alexa Fluor 594 [1:500; Life Technologies], and anti-mouse IgG [H+L] Alexa Fluor 647 [1:500; Life Technologies]) and Hoechst-33342 (5 µg/ mL; Sigma-Aldrich) in PBS-B-T buffer for 60 min at room temperature.