Sc 32892

The cells were fixed with paraformaldehyde, blocked with 1% BSA, incubated with primary antibodies overnight at 4°C, reacted with the corresponding secondary antibodies for 2 hours at 37°C the next day, then stained the nucleus with 40,6-diamino-2-phenylindole (DAPI; Sigma-Aldrich) for 5 minutes, and observed using the confocal fluorescence microscope. Antibodies used included CK14 (1 : 200, ab49747, Abcam), vimentin (1 : 200, OMA1-06001, Thermo), Sox2 (1 : 200, ab97959, Abcam), AMBN (1 : 200, sc50534, Santa Cruz), and AMGN (1: 200, sc32892, Santa Cruz).

Paraffin-embedded samples were sectioned at a thickness of 5 μm. Selected sections were then stained with haematoxylin and eosin (H&E) and analysed under bright-field microscopy (BX53; Olympus, Tokyo, Japan). For Masson's trichrome staining, we used Weigert's iron haematoxylin, Biebrich scarlet-acid fuchsin and aniline blue. Immunohistochemical analyses were performed on selected sections of each construct using a tooth-specific antibody against amelogenin (sc-32892; Santa Cruz Biotechnology, Paso Robles, CA, USA), a TGF-β pathway-specific antibody against p-Smad2/3 (sc-11769; Santa Cruz Biotechnology, Paso Robles, CA, USA) and an antibody against smad2/3 (NBP1-19520; Novus Biologicals, Littleton, CO, USA). For immunohistochemistry (IHC), all sections were incubated with a biotinylated secondary antibody, stained using the R&D HRP-DAB staining kit (R&D Systems, Minneapolis, MN, USA) and counterstained with haematoxylin. Processed sections were dehydrated through a series of graded ethanol baths, sealed with Permount (Thermo Fisher Scientific, Waltham, MA, USA), and analysed using bright-field microscopy. Photographs were obtained with a digital camera and manipulated using Adobe Photoshop.