2.6.8.Immunohistochemical (IHC) analysis
Deciduous molars were separated and preserved in 10% neutral
formaldehyde. IHC analysis was performed as previously
described[12]. The rabbit anti-AFM,
anti-GJA1, anti-PAX9, and anti-KRT15 primary antibodies were purchased
from PTM Bio. Briefly, the peraffin-embedded sections were blocked with
3% hydrogen dioxide for 30 min and retrieved by autoclaving in citrate
repair buffer for 2 min. After 3 times of phosphate-buffered saline
(PBS) washing, the sections were incubated with primary antibodies at
the temperature of 4 ℃ overnight. Two-Step IHC reagents (Zhongshan
Goldenbridge, China) were used for IHC staining. Hematoxylin was used
for counterstaining. Finally, at least 10 randomly selected images with
high-resolution fields were analyzed using Image-Pro Plus version 6.0.