Ab9542

DPSCs and SHED were cultured on coverslips coated with poly-L-lysin. After washing with PBS (Invitrogen), the samples were fixed with 4% paraformaldehyde. The fixed cells were permeabilized with PBS containing 0.25% Triton X-100 (PBST; Bio Basic, Seoul, Korea), washed, and incubated with 5% BSA (Sigma–Aldrich) in PBST to block nonspecific antibody binding.
The cells were incubated in primary antibodies diluted in 5% BSA/PBST overnight at 4 °C. The primary antibodies included antihuman interleukin-6 (IL-6; rabbit polyclonal antibody, ab6672, Abcam; 1:500), antihuman brain-derived neurotrophic factor (BDNF; rabbit monoclonal antibody, ab108319, Abcam; 1:200), antihuman placental growth factor (PLGF; rabbit polyclonal antibody, ab9542, Abcam; 1:500), and antihuman vascular endothelial growth factor D (VEGF-D; rabbit monoclonal antibody, ab155288, Abcam; 1:200). After washing, the cells were incubated with biotinylated secondary antibody (biotinylated antirabbit IgG, Vector Labs, Burlingame, CA, USA) in 5% BSA/PBST for 1 h at RT. After washing, the cells were incubated with streptavidin–HRP (Vector Labs) in 5% BSA/PBST for 30 min at RT. Color was developed using 3,3ʹ-diaminobenzidine substrate (Abcam) and hematoxylin (Merck). Coverslips were mounted using a drop of mounting medium (Vector Labs) and stored at RT.