A Nikon U-2000 microscope was used to collect the immunofluorescent images. The expression of F-actin, Collagen-I and β1-integrin were tracked at 6-32 h post-plating. The expression of β3-tublin and Neu-N were examined in cells at Day 1 and Day 5 of differentiation. The primary antibodies used in this study include mouse anti-F-actin (Millipore, Temecula CA, 1:100 dilution), rabbit anti-collagen-I (Abcam, Cambridge, MA 1:100 dilution), rabbit anti-β3-Tublin (Tuj1, Abcam, 1:200 dilution), mouse anti-NeuN (Millipore, 1:100 dilution) and rabbit anti-β1-integrin (Santa Cruz Biotechnology, 1:100 dilution). Secondary antibodies were purchased from Invitrogen (Carlsbad, CA) and used at a dilution of 1:200.
The images were quantitatively analyzed by ImageJ (NIH). In determining the percentage of positively stained cells, we set the threshold at 50% of the highest staining intensity across the samples. Cell length-to-width ratio was analyzed to reveal cell polarization. Since cell width varies along an elongated cell, average cell width was evaluated from the ratio of cell area and cell length.
The images were quantitatively analyzed by ImageJ (NIH). In determining the percentage of positively stained cells, we set the threshold at 50% of the highest staining intensity across the samples. Cell length-to-width ratio was analyzed to reveal cell polarization. Since cell width varies along an elongated cell, average cell width was evaluated from the ratio of cell area and cell length.