Biorp

Cells cultured on coverslips were fixed with 4% paraformaldehyde at 4 °C for 30 min. After washing with PBS, cells were permeabilized in PBS containing 1% Triton X-100 and 0.5% NP-40 (Sigma-Aldrich) and then blocked with 3% bovine serum albumin (BSA) and 1% normal horse serum (Santa Cruz Biotechnology). Cells were incubated with antibodies against Tuj1 (1:100, Abcam, Cambridge, UK), cleaved caspase3 (1:100, Cell Signaling Technology), and then appropriate secondary antibodies (Invitrogen). Cells were stained with a TUNEL kit (Roche, Indianapolis, IN, USA), according to the manufacturer’s protocol. 4′,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories, Burlingame, CA, USA) was used for the nuclei. After mounting, the cells were examined using confocal laser scanning microscopy (BIORP, Leica, Wetzlar, Germany).

Cells were fixed in ice-cold 4% paraformaldehyde (PFA, Biosesang, Gyeonggi, South Korea) at room temperature (RT) for 20 min, permeabilized in 0.2% Triton X-100 for 20 min, blocked using blocking buffer containing 2% normal goat serum (NGS, Abcam) and 1% bovine serum albumin (BSA, GenDEPOT, Barker, TX, USA) in PBS, and then incubated with the following primary antibodies: Nestin (1:200, Novus Biologicals, Littleton, CO, USA); Ki67 (1:250, Abcam, Cambridge, MA, USA); Tuj1 (1:1000, Abcam); GFAP (1:2000, Abcam), Claudin11 (1:250, Abcam) at 4 °C overnight. Samples were rinsed and then treated with Alexa Fluor 594-conjugated goat anti-Rabbit IgG antibodies (1:500, Abcam) and the nuclei were counterstained with DAPI for 10 min at RT. After mounting, the cells were examined using confocal laser scanning microscopy (BIORP, Leica, Wetzlar, Germany).