Mandible Analysis: Enamel Protein IHC

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.