Anti runx3

At 1, 4, 7, and 14 days post-surgery, nerve samples from proximal nerve stump and normal nerve sections (0 day) were obtained for cutting into longitudinal sections that were subjected to immunoffuorescent triple-staining with rabbit anti-ALCAM polyclonal antibody (1:100 dilution, Abcam), anti-CREB1 (1:800 dilution, CST), anti-NRP1 (1:200 dilution, Abcam), anti-NRP2 (1:100 dilution, CST), anti-RAC1 (1:100 dilution, Sigma), anti-RUNX3 (1:600 dilution, Abcam), mouse anti-NF200 (1:400 dilution, Sigma) and Hoechst 33342 (1:5,000 dilution, Life Technologies) respectively. Primary antibodies incubated with the nerve sections at 4°C overnight, followed by further reaction with the secondary antibody (Goat anti-Mouse IgG-Alex-488, 1:500 and Donkey anti-Rabbit IgG-Cy3, 1: 1,000) at 4°C overnight, and nerve sections were observed by the aid of a confocal laser scanning microscope (TCS SP2, Leica).

Cells were treated with PDT, incubated for 4 h, then lysed (1.5×10⁵ cells) in cell lysis buffer (Cell Signaling Technology, MA, USA). Next, proteins were separated using 10% SDS-PAGE and transferred to a methanol-activated polyvinylidene fluoride membrane (Bio-Rad, CA, USA). The membrane was blocked for 1 h in PBST containing 5% milk and subsequently probed with anti-cleaved caspase-3 antibody, anti-cleaved caspase-9 antibody (Cell Signaling Technology, MA, USA), anti-RUNX3 (1:1,000) was obtained from Abcam (Abcam, Cambridge, UK). Additionally, antibodies against Bax (1:1,000), Bim (1:1,000) and Bcl-2 (1:1,000) were obtained from BioVision (BioVision, CA, USA). An anti-actin antibody (1:2,000) was used as a loading control (Sigma, MO, USA). Blots were then incubated for 1 h with horseradish peroxidase-conjugated anti-mouse (1:2,000) or rabbit (1:2,000) secondary antibodies (Cell Signaling Technology, MA, USA), and detection was done by chemiluminescence (Santa Cruz Biotechnology, CA, USA). The density was detected using chemiluminescence (Santa Cruz Biotechnology, CA, USA).