Sc 376177

hNPCs were cultured on laminin-coated 13 mm glass coverslips, and the cells were fixed post HG treatment using 4% paraformaldehyde (PFA) for 18 min at room temperature, and then blocked with 5% goat serum for 1 h. Subsequently, the cells were incubated at 4 °C overnight with rabbit anti-SLIT1 (1:100, ab129345, Abcam), mouse anti-ROBO2 (1:200, sc-376177, Santa Cruz Biotechnology), mouse anti-SRGAP1 (1:200, sc-81939, Santa Cruz Biotechnology), mouse anti-YAP (1:50, sc-101199, Santa Cruz Biotechnology), rabbit anti- TAZ (1:50, ab84927, Abcam), rabbit anti-CDC42 (1:50, #2462, Cell Signaling Technology), mouse anti-Nestin (1:250, ab18102, Abcam), mouse anti-SOX-2 (1:250, ab171380, Abcam) or rabbit anti-Musashi-1 (1:500, ab52865, Abcam). The following day, the coverslips were incubated with secondary antibody for 1 h at room temperature. The nucleus was counterstained with 4′, 6-diamidino-2-phenylindole (DAPI) for 5 min, and the coverslips were mounted with fluorescence mounting media (DAKO, Cat. no. S3023) onto glass slides. Images were taken using a confocal microscope, LSM FV1000 (Olympus).

The fixed mouse sections were permeabilized with 0.1% PBS-Tx for 10 min, following which sections were washed twice with PBS for 10 min each. The sections were then blocked with 5% goat serum for 1 h. Next, the sections were incubated with rabbit anti-SLIT1 (1:25, ab129345, Abcam), mouse anti-ROBO2 (1:100, sc-376177, Santa Cruz Biotechnology), mouse anti-SRGAP1 (1:50, sc-81939, Santa Cruz Biotechnology) or rabbit anti-CDC42 (1:25, #2462, Cell Signaling Technology), overnight. The following day, the sections were washed with PBS thrice for 10 min each before incubation with secondary antibody for 1 h. Next, the sections were washed thrice with PBS thrice for 10 min each, and the nucleus was counterstained with DAPI. The coverslips were mounted using mounting medium (DAKO) and imaged using a confocal microscope (Olympus FV1000).