Immunofluorescence Characterization of Dental Pulp Stem Cell Differentiation

The expression of proliferation (e.g., transient receptor potential canonical 1, TRPC1), stemness (e.g., CD146), and neuronal differentiation (e.g., Nestin and MAP-2) markers was determined in d-DPSCs cultured in the different media by immunofluorescence staining as described previously (Luo et al., 2018a (link)). At the designated time points after culture, the cells were fixed in 4% paraformaldehyde for 30 min and incubated for 30 min in a blocking solution containing 5% BSA (Amresco) and 0.1% Triton X-100 (Solarbio) at room temperature. The cells were incubated with the following primary antibodies at 4°C overnight: anti-TRPC1 (1:50; Santa Cruz Biotechnology), anti-CD146 (1:200; Abcam), anti-Nestin (1:1000; Sigma-Aldrich), and anti-MAP-2 (1:500, Sigma-Aldrich). The secondary antibodies were donkey anti-rabbit IgG H&L and donkey anti-mouse IgG H&L (1:500; Abcam). Cell nuclei were stained with DAPI (Beyotime), and images were taken by fluorescence microscopy (Eclipse 80i; Nikon, Japan). Both the culture of undifferentiated DPSCs in complete α-MEM containing 10% FBS for 6 days and the culture of DPSCs in continuous neuronal differentiation medium for 12 days (the neural-induction group) served as control groups.