Bis benzamide dapi

Human NPCs were characterized by immunocytochemistry. Briefly, cells grown on glass coverslips were fixed with 4% paraformaldehyde and incubated in the blocking buffer (5% goat serum, 1% bovine serum albumin, and 0.1% Triton X-100) for 15 min. Cells were then incubated in primary antibodies diluted in the blocking buffer at 4 °C overnight. Appropriate secondary antibodies were used for single and double labeling. All secondary antibodies were tested for cross-reactivity and nonspecific immunoreactivity. The following primary antibodies were used, anti-SOX2 (1:200, R&D Systems), anti-Nestin (1:500, R&D Systems), anti-SOX1 (1:250, Millipore). Bis-benzamide (DAPI, 1:1000; Sigma) was used to visualize the nuclei. Images were captured using a Zeiss Axiovision microscope with z-stack split view function.

The undifferentiated and differentiated Nestin+ cells were characterized by immunocytochemistry [26 (link),27 (link)]. Briefly, cells grown on glass coverslips were fixed with 2% paraformaldehyde for 10 min at 4 °C and then washed 3 times in PBS. The fixed cells were incubated in blocking buffer (PBS containing 5% goat serum, 1% bovine serum albumin, and 0.1% Triton X-100) for 30 min at room temperature (RT) and were then followed in blocking buffer containing primary antibodies at the indicated concentrations overnight at 4 °C. Appropriate secondary antibodies were used for single and double labeling. All secondary antibodies were tested for cross-reactivity and nonspecific immunoreactivity. The following primary antibodies were used: Nestin (1:200, Abcam, Waltham, MA, USA), Sox2 (1:200, R&D Systems, Minneapolis, MN, USA), SOX1 (1:200, R&D Systems), OCT 4 (1:500, Abcam), SSEA4 (1:10, Development studies hybridoma Bank, DSHB, Iowa City, IA, USA), GFAP (1:4000, DAKO/Agilent, Carpinteria, CA, USA), tubulin βIII (1:1000, Sigma, St. Louis, MO, USA), MAP2 (1:200, Sigma), human nuclei antigen (hN) (1:200, Millipore, Burlington, MA, USA), NeuN, (1:200, Sigma), and neurofilament-light chain (NFL) (1:200, Sigma). Bis-benzamide (DAPI, 1:1000; Sigma) was used to visualize the nuclei. Images were captured using a Zeiss Axiovision microscope with z-stack split view function.