Assessing Ameloblast Barrier Function

The apical tight junction is an important indicator for ameloblast polarity and can be measured by the functional selectivity of the paracellular apical barrier. Sulfo-NHS-Biotin was used as a tracer molecule to evaluate the effects of SATB1 on the ameloblast layer barrier function as previously described [78 (link)]. Briefly, four 10-day old pups from each wt and Satb1^−/− mouse model were anesthetized by intraperitoneal injection with 0.05 ml/10 g of ketamine (18 mg/ml). A 30-G injection needle with a blunt end was carefully inserted into the left ventricle of the heart, and 150 μl/g of Sulfo-NHS-Biotin solution was perfused into the bloodstream. Ten minutes after receiving Sulfo-NHS-Biotin injection, the pups were sacrificed, and the hemimandibles were dissected, processed, and paraffin-embedded for histological sectioning as previously mentioned. The sagittal sections were blocked with 5% BSA for 1 h then incubated with alkaline phosphatase-conjugated streptavidin (Vector Laboratories, Inc.) for 30 min, and immunoreactivity was visualized using a Vector® Red kit (Vector Laboratories, Inc.).