Sc 50534

The first lower molars dissected from 4-day-old WT, ENAM^Rgsc514 heterozygous, and ENAM^Rgsc514 homozygous mice were ground into powder in liquid nitrogen and lysed using RIPA buffer (ThermoFisher Scientific, Waltham, MA, USA) containing a proteinase inhibitor cocktail (Roche, Indianapolis, IN, USA). After quantitation by bicinchoninic acid (BCA) protein assay (Thermo Fisher Scientific), the lysates containing equal amounts of total proteins from each group were loaded on sodium dodecyl sulfate polyacrylamide gel (SDS–PAGE) and analyzed by Western immunoblotting using the antibodies anti-ENAMN-terminus (1:1 600),¹³ anti-AMBN C-terminus (1:2 000, SC-50534, Santa Cruz Biotechnology), anti-AMEL (1:2 000, SC-32892, Santa Cruz Biotechnology), and β-ACTIN(1:3 000, SC-47778, Santa Cruz Biotechnology), using the methods described previously.^{6 (link), 14 (link), 15 (link), 16 (link)}

The mandibles collected from 6-day-old mice were fixed in 4% paraformaldehyde at 4 °C for 16 h and then decalcified in 8% ethylene diamine tetraacetic acid (EDTA)/PBS (pH 7.4) at 4 °C for 4 days, followed by paraffin embedding. Five-μm thick serial sections were prepared for H&E and immunohistochemistry (IHC) staining, as we previously described.^{8 (link)} The primary antibodies used for IHC staining were: anti-ENAM N-terminus (1:600),¹³ anti-ENAM C-terminus (1:50, SC-33107, Santa Cruz Biotechnology, CA, USA), anti-AMBN N-terminus (1:50, SC-33100, Santa Cruz Biotechnology), anti-AMBN C-terminus (1:600, SC-50534, Santa Cruz Biotechnology), and anti-AMEL (1:600, SC-32892, Santa Cruz Biotechnology). Methyl green was used for the counterstaining of IHC analyses.

Offspring were collected at E15.5, E17.5, and P0.5. Paraffin sections were prepared and immunostaining was performed as previously described23 (link). Primary antibodies against Ki67 (ab15580, Abcam), AMBN (sc-50534, Santa Cruz), AMGN (sc-32892, Santa Cruz), and APEX1 (ab194, Abcam) were used. To determine the cell proliferation index, Ki67-positive cells in the cervical loop of each tissue section were counted. At least five serial sections from each animal were examined, and three animals from different litters (n = 3) were included in each group.