Nestin antibody

All samples were embedded in paraffin blocks and sectioned longitudinally. H&E was performed to evaluate the morphology and structure of the regenerated tissues. To evaluate the neovascularization in the transplants, three sections stained with H&E were captured using an optical microscope (Olympus) at ×4, ×10, and ×40 magnification. In addition, a red fluorescence-labeled CD31 antibody (Santa Cruz) was used to detect the ECs. Images were taken at ×20 magnification. The areas of vessels in H&E images and CD31⁺ cells-lined lumens in immunofluorescent images were respectively quantified by ImageJ. Human-specific DSPP antibody (Santa Cruz), DMP-1 antibody (Biovision, Milpitas, CA), and Nestin antibody (Santa Cruz) were detected to confirm the odontoblastic differentiation of the migrated h-DPSCs by immunohistochemistry. Human-specific mitochondria antibody (Abcam) was detected to determine whether the regenerated pulp-like tissue is host-derived or donor-derived using immunohistochemistry and immunofluorescence. The skin tissue was used for negative control.

The cells were fixed with 4% paraformaldehyde (PFA) for 30 min at 4°C, and then washed in cold PBS for 5 min 3 times. The nuclei were subsequently permeabilized with PBS with 0.5% Triton X‐100 for 30 min. Next, the cells were blocked with 1% BSA in PBS for 1 h. The cells were incubated with primary antibody overnight at 4°C. Primary antibodies used were cleaved CASPASE 3 antibody (9664S, CST), Ki67 antibody (ab15580, Abcam), NESTIN antibody (Santa Cruz, sc‐58813), NR2E1 antibody (Santa Cruz, sc‐377240X), GFAP antibody (Millipore, 5541) and TUJ‐1 (abcam, ab1445). After wash, the cells were incubated with anti‐rabbit secondary antibody conjugated with proper Alexa Fluor label for 1 h at room temperature in darkness. The nuclei were counterstained with DAPI. The cells were imaged with Olympus IX‐73 immunofluorescence microscope.