Furthermore, an immunocytochemistry study was performed using cultured dental epithelial mDE6 cells to confirm the intracellular localization of Itm2a protein in the dental epithelial cells. We herein address the possible functional roles of Itm2a during tooth development based on these results. An immunofluorescent histochemical analysis was also carried out using an anti-Itm2a antibody. On E10.5 and E12, Itm2a protein JNJ-42041935 expression was not detected in the epithelial or mesenchymal cells corresponding to the predicted lower first molar region, nor was Itm2a mRNA. At the bud stage, Itm2a protein expression was not observed in the epithelial cells of the tooth bud or in the mesenchymal cells condensed around the tooth bud. However, the protein expression of Itm2a in the subsequent stages was detected in the developing tooth germ, and demonstrated a similar expression pattern to that of the mRNA. Some differences were identified, including a time lag, between the mRNA and protein expression, as indicated in the respective regions below. Immunofluorescent signals for the Itm2a protein were observed in the enamel organ on E15 and on the subsequent days, as was the ML-098 signal for Itm2a mRNA. Although a signal for the mRNA was observed in the PEK on E15, a signal for the protein was undetectable in the PEK at that time. In the inner enamel epithelium, the signal for the protein became clearer on E16 after the mRNA signal was detected. On E17-PN0, a protein signal was detected in the inner enamel epithelium, but the intensity in the region near the cervical loop was weaker than that in the other regions at the presumptive cusp sites. A strong protein signal for Itm2a in the ameloblasts and preameloblasts on PN1 and on the subsequent days demonstrated similar expression patterns to that of the mRNA signal observed on these days. The outer enamel epithelium showed an immunofluorescent signal for protein expression on E15 that was similar to that of the mRNA expression. On E16, the signal intensity appeared to be increased on the buccal side. On E17, the signal was clearly observed on both the buccal and lingual sides.